Compositions and methods for the therapy and diagnosis of inflammatory bowel disease

ABSTRACT

Compositions and methods for the therapy and diagnosis of Inflammatory Bowel Disease (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, are disclosed. Illustrative compositions comprise one or more bacterial polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of IBD.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation in part of PCT Application No.PCT/US02/40422, filed on Dec. 16, 2002, which is related to U.S.Provisional Patent Application No. 60/426,835, filed Nov. 15, 2002, No.60/396,242, filed Jul. 16, 2002, and No. 60/341,830, filed Dec. 17,2001, which are all incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to therapy and diagnosisof Crohn's Disease and Ulcerative Colitis (collectively referred to asInflammatory Bowel Disease, or IBD). The invention is more particularlyrelated to polypeptides comprising at least a portion of a protein thatis recognized, and to which individuals with IBD mount an aberrantimmune response, and to polynucleotides encoding such polypeptides. Suchpolypeptides and polynucleotides are useful in pharmaceuticalcompositions, e.g., vaccines, and other compositions for the diagnosisand treatment of IBD.

[0004] 2. Description of the Related Art

[0005] Crohn's Disease and Ulcerative Colitis (collectively referred toas Inflammatory Bowel Disease, or IBD) are chronic, inflammatorydiseases of the gastrointestinal tract. While the clinical features varysomewhat between these two disorders, both are characterized byabdominal pain, diarrhea (often bloody), a variable group of‘extra-intestinal’ manifestations (such as arthritis, uveitis, skinchanges, etc) and the accumulation of inflammatory cells within thesmall intestine and colon (observed in pathologic biopsy or surgicalspecimens).

[0006] IBD affects both children and adults, and has a bimodal agedistribution (one peak around 20, and a second around 40). IBD is achronic, lifelong disease, and is often grouped with other so-called“autoimmune” disorders (e.g. rheumatoid arthritis, type I diabetesmellitus, multiple sclerosis, etc). IBD is found almost exclusively inthe industrialized world. The most recent data from the Mayo Clinicsuggest an overall incidence greater than 1 in 100,000 people in theUnited States, with prevalence data in some studies greater than 1 in1000. There is a clear trend towards the increasing incidence of IBD inthe US and Europe, particularly Crohn's Disease. The basis for thisincrease is not presently clear. As such, IBD represents the 2^(nd) mostcommon autoimmune disease in the United States (after rheumatoidarthritis).

[0007] Treatment of IBD is varied. First line therapy typically includessalicylate derivatives (e.g. 5-ASA) given orally or rectally. Responserates in uncomplicated Crohn's Disease are approximately 40% (comparedto 20% for placebo). Corticosteroids remain a mainstay in the treatmentof patients with more “refractory” disease, despite the untowardside-effects. Newer treatment options include anti-metabolites (e.g.methotrexate, 6-mercaptopurine) and immunomodulators (e.g. Remicade—achimeric human antibody directed at the TNFα receptor).

[0008] In spite of considerable research into therapies for thesedisorders, IBD remains difficult to diagnose and treat effectively.Furthermore, there are no clear laboratory tests that are diagnostic forIBD, nor are there suitable laboratory tests that serve as “surrogatemarker” that are uniformly useful to follow the course of disease inpatients. Accordingly, there is a need in the art for improved methodsfor detecting and treating such inflammatory bowel diseases. The presentinvention fulfills these needs and further provides other relatedadvantages.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIG. 1 shows a schematic of flagellin clones with percentsimilarity to related flagellin B from the rumen anaerobe, Butyrivibriofibrisolvens.

[0010]FIG. 2a shows Western Blot analysis of serum antibody response torecombinant flagellins cBir-1 and Fla^(X) and fragments.

[0011]FIG. 2b shows titration of serum anti-flagellin antibody againstrecombinant flagellins cBir-1 and Fla^(X).

[0012]FIG. 2c shows the correlation of colitis score with serumanti-Fla^(X).

[0013]FIG. 2d shows association of anti-flagellin antibody with humaninflammatory bowel diseases. NL is negative population, UC is ulcerativecolitis positive, IC is inflammatory control, CD is Crohn's Diseasepositive and UC+ and CD+ pANCA+ are ulcerative colitis pANCA positiveand Crohn's Disease pANCA positive.

[0014]FIGS. 3a and b show cytokine release by donors stimulated withflagellin.

BRIEF SUMMARY OF THE INVENTION

[0015] In one aspect, the present invention provides polynucleotidecompositions comprising a sequence selected from the group consistingof:

[0016] (a) sequences provided in SEQ ID NOs: 75, 83, 85, 1-37, 51-74,76-78 and 87;

[0017] (b) complements of the sequences provided in SEQ ID NOs: 75, 83,85, 1-37, 51-74, 76-78 and 87;

[0018] (c) sequences consisting of at least 20, 25, 30, 35, 40, 45, 50,75 and 100 contiguous residues of a sequence provided in SEQ ID NOs: 75,83, 85, 1-37, 51-74, 76-78 and 87;

[0019] (d) sequences that hybridize to a sequence provided in SEQ IDNOs: 75, 83, 85, 1-37, 51-74, and 76-78, under moderate or highlystringent conditions;

[0020] (e) sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identity to a sequence of SEQ ID NOs: 75, 83, 85, 1-37,51-74, 76-78 and 87;

[0021] (f) degenerate variants of a sequence provided in SEQ ID NOs: 75,83, 85, 1-37, 51-74, 76-78 and 87.

[0022] The present invention, in another aspect, provides polypeptidecompositions comprising an amino acid sequence that is encoded by apolynucleotide sequence described above.

[0023] The present invention further provides polypeptide compositionscomprising an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NOs: 79, 84, 86, 80-82, 38-50 and 88-89.

[0024] In certain preferred embodiments, the polypeptides and/orpolynucleotides of the present invention are immunogenic, i.e., they arecapable of eliciting an immune response, particularly a humoral and/orcellular immune response, as further described herein.

[0025] The present invention further provides fragments, variants and/orderivatives of the disclosed polypeptide and/or polynucleotidesequences, wherein the fragments, variants and/or derivatives preferablyhave a level of immunogenic activity of at least about 50%, preferablyat least about 70% and more preferably at least about 90% of the levelof immunogenic activity of a polypeptide sequence set forth in SEQ IDNOs: 79, 84, 86, 80-82, and 38-50 or a polypeptide sequence encoded by apolynucleotide sequence set forth in SEQ ID NOs: 75, 83, 85, 1-37,51-74, 76-78 and 88-89.

[0026] The present invention further provides polynucleotides thatencode a polypeptide described above, expression vectors comprising suchpolynucleotides and host cells transformed or transfected with suchexpression vectors.

[0027] Another aspect of the present invention provides for isolatedantibodies, or antigen-binding fragment thereof, that specifically bindto the polypeptides of the present invention. In one embodiment of theinvention, the antibody may be a monoclonal antibody. In a furtherembodiment the antibody is a human antibody or an antibody that has beenhumanized. In yet further embodiments, the antibodies of the presentinvention bind to flagellin proteins and in one embodiment theantibodies are neutralizing antibodies against flagellin proteins. In anadditional embodiment, said antibodies block the interaction between aflagellin protein and a Toll-like receptor. In one particularembodiment, the Toll-like receptor is TLR5.

[0028] The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

[0029] The present invention also provides, in other aspects,oligonucleotides that hybridize to the polynucloeotides of the presentinvention. In one embodiment, the oligonucleotides hybridize to thepolynucleotides of the present invention under highly stringentconditions. In one embodiment, the oligonucleotides hybridize topolynucleotides that encode flagellin proteins.

[0030] The present invention further provides, in one aspect, methods ofstimulating and/or expanding T cells specific for an enteric bacterialprotein, comprising contacting T cells with at least one componentincluding but not limited to, polypeptides or polynucleotides of thepresent invention, antigen-presenting cells that express apolynucleotide of the present invention under conditions and for a timesufficient to permit the stimulation and/or expansion of T cells. In oneembodiment of the invention the T cells are CD4+ T cells. In a furtherembodiment, the CD4+ T cells mediate a decrease in inflammation in thecolon. In another embodiment the T cells are specific for a flagellinpolypeptide.

[0031] The present invention, in one aspect, also provides forpopulations of T cells produced according to the methods describedherein. In one embodiment, said T cells produce cytokines that mayinclude, but are not limited to, interleukin 10 (IL-10), interferon-β(IFN-β), interleukin 4 (IL-4), interleukin 12 (IL-12), transforminggrowth factor beta (TGFβ or interleukin 18 (IL-18). In preferredembodiments, the T cells produce IL-10 and/or TGFβ.

[0032] Within other aspects, the present invention providespharmaceutical compositions comprising a polypeptide or polynucleotideas described above and a physiologically acceptable carrier.

[0033] Within a related aspect of the present invention, thepharmaceutical compositions, e.g., vaccine compositions, are providedfor prophylactic or therapeutic applications. Such compositionsgenerally comprise an immunogenic polypeptide or polynucleotide of theinvention and an immunostimulant, such as an adjuvant.

[0034] The present invention further provides pharmaceuticalcompositions that comprise: (a) an antibody or antigen-binding fragmentthereof that specifically binds to a polypeptide of the presentinvention, or a fragment thereof; and (b) a physiologically acceptablecarrier.

[0035] The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

[0036] Within further aspects, the present invention providespharmaceutical compositions comprising: (a) T cells specific for apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Illustrative T cells include T cells expressing avariety of cytokines including interleukin 10 (IL-10), interferon-β(IFN-β), interleukin 4 (IL-4), interleukin 12 (IL-12), transforminggrowth factor beta (TGFβ or interleukin 18 (IL-18). In preferredembodiments, the T cells produce IL-10 and/or TGFβ.

[0037] Within further aspects, the present invention providespharmaceutical compositions comprising: (a) an antigen presenting cellthat expresses a polypeptide as described above and (b) apharmaceutically acceptable carrier or excipient. Illustrative antigenpresenting cells include dendritic cells, macrophages, monocytes,fibroblasts and B cells.

[0038] Within related aspects, pharmaceutical compositions are providedthat comprise: (a) T cells specific for a polypeptide as described aboveor an antigen presenting cell that expresses a polypeptide as describedabove and (b) an immunostimulant. Illustrative immunostimulants includeadjuvants such as Freund's Incomplete Adjuvant; Freund's CompleteAdjuvant; Merck Adjuvant 65; AS-1, AS-2; aluminum hydroxide gel;aluminum phosphate; a salt of calcium, iron or zinc; an insolublesuspension of acylated tyrosine acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A, QS21, aminoalkyl glucosaminide4-phosphates, or quil A.

[0039] In a related aspect, the present invention provides a method ofstimulating an immune response in a mammal, comprising administering tothe mammal the compositions described above. In one embodiment, theimmune response comprises T cells that produce a cytokine including, butnot limited to, interleukin 10 (IL-10), interferon-β (IFN-β),interleukin 4 (IL-4), interleukin 12 (IL-12), transforming growth factorbeta (TGFβ or interleukin 18 (L-18). Particularly illustrative cytokinescomprise IL-10 and/or TGFβ.

[0040] In a related aspect, the present invention provides a method ofdecreasing gastrointestinal inflammation associated with inflammatorybowel disease in a mammal, comprising administering to said mammal thecompositions of the present invention.

[0041] Another aspect of the present invention provides for a method ofdetecting the presence of inflammatory bowel disease in a mammalcomprising contacting a biological sample from the mammal, wherein saidbiological sample comprises antibodies, with the polypeptides describedabove, detecting in the sample an amount of antibody that binds to thepolypeptide; and comparing the amount of bound antibody to apredetermined cut-off value and therefrom determining the presence ofinflammatory bowel disease in the mammal. Illustrative biologicalsamples include sera, stool, tissue or other material obtained bycolonoscopy, ileoscopy, esophagogastroduodenoscopy (EGP), or surgery. Inone particular embodiment the polypeptide comprises a flagellinpolypeptide.

[0042] Another aspect of the present invention provides for a method ofdetecting the presence of inflammatory bowel disease in a mammalcomprising contacting a biological sample from the mammal, wherein saidbiological sample comprises polynucleotides, with at least oneoligonucleotide that is at least in part complementary to apolynucleotide described above, detecting in the sample an amount of apolynucleotide that hybridizes to said oligonucleotide; and comparingthe amount of said polynucleotide that hybridizes to saidoligonucleotide to a predetermined cut-off value, and therefromdetermining the presence of inflammatory bowel disease in a mammal. Inone embodiment, the oligonucleotide hybridizes under moderatelystringent conditions. In a particular embodiment, the polymerase chainreaction is used to determine the amount of polynucleotide thathybridizes to said oligonucleotide. In another embodiment, ahybridization assay is used to determine the amount of polynucleotidethat hybridizes to said oligonucleotide. Illustrative biological samplescomprising polynucleotides include sera, stool, tissue or other materialobtained by colonoscopy or colonic biopsy, ileoscopy,esophagogastroduodenoscopy (EGP), or surgery. In one particularembodiment, the polynucleotide encodes a flagellin protein.

[0043] Another aspect of the present invention provides for a method ofstimulating and/or expanding B cells that produce antibodies specificfor an enteric bacterial protein, comprising contacting B cells with thepolypeptides or polynucleotides mentioned above under conditions and fora time sufficient to permit the stimulation and/or expansion of B cells.In one embodiment the B cells produce antibodies that bind to aflagellin protein. In another embodiment said antibodies areneutralizing antibodies against a flagellin protein. In anotherembodiment, the antibodies block the interaction between a flagellinprotein and a Toll-like receptor. In one particular embodiment, theToll-like receptor is TLR5.

[0044] Within related aspects, the present invention provides forpopulations of B cells generated as described above.

[0045] Within further aspects, the present invention provides a methodof identifying bacterial antigens associated with inflammatory boweldisease in a mammal, comprising contacting a biological sample thatcomprises T cells with the polynucleotides, or polypeptides describedabove, or antigen-presenting cells that express a polynucleotidedescribed herein, under conditions and for a time sufficient to permitthe stimulation and/or expansion of T cells, and further, detecting inthe sample the magnitude of said stimulation and/or expansion of Tcells; and, comparing the magnitude of said stimulation and/or expansionto a predetermined cut-off value, and therefrom identifying bacterialantigens associated with inflammatory bowel disease in the mammal. Inone embodiment, the mammal is a human. In a further embodiment themammal is a mouse. Illustrative mouse strains are C3H/HeJ Bir, BALB/cIL-10−/−, B6 IL-10−/−, B10 IL-10−/−, MDR1a −/−, TCRα−/−, IL-2−/−, IL-2R−/−, mice with DSS (Dextransodiumsulfate) induced colitis, Gα_(ai) −/−,and CD45 RB transgenic mice. In one particular embodiment, the strain ofmouse is C3H/HeJ Bir. In another embodiment, the mammal is a rat. In oneparticular embodiment, the rat is an HLA-B27-transgenic rat.

[0046] In certain other aspects, the present invention provides methodsof monitoring the progression of inflammatory bowel disease in a mammal,comprising the steps of: (a) obtaining a biological sample from themammal, wherein said biological sample comprises antibodies; (b)contacting the biological sample with a polypeptide described herein;(c) detecting in the sample an amount of antibody that binds to thepolypeptide; and (d) repeating steps (a), (b), and (c) using abiological sample obtained from the mammal at a subsequent point intime; and (e) comparing the amount of bound antibody in step (c) to theamount of bound antibody in step (d) and therefrom monitoring theprogression of inflammatory bowel disease in the mammal.

[0047] Another aspect of the present invention provides methods ofidentifying bacterial antigens associated with inflammatory boweldisease in a first mammal, comprising the steps of: (a) obtaining abiological sample from said first mammal wherein said biological samplecomprises DNA from cecal bacteria; (b) constructing an expressionlibrary with said DNA; (c) screening said expression library with serafrom either said first mammal or a second mammal with inflammatory boweldisease; thereby identifying bacterial antigens associated withinflammatory bowel disease. In one embodiment, both the first and secondmammals are mice. In a related embodiment, said first mammal is aC3H/HeJ Bir mouse and said second mammal is a different strain of mouseincluding BALB/c IL-10−/−, B6 IL-10−/−, MDR1a −/−, or CD45 RB transgenicmice. In another related embodiment, said first mammal is a mouse andsaid second mammal is a human.

[0048] In certain other aspects, the present invention provides methodsof monitoring the progression of inflammatory bowel disease in a mammal,comprising the steps of (a) obtaining a biological sample from saidmammal, wherein said biological sample comprises polynucleotides; (b)contacting said sample with at least one oligonucleotide that is atleast in part complementary to a polynucleotide described herein; (c)detecting in the sample an amount of a polynucleotide that hybridizes tosaid oligonucleotide; (d) repeating steps (a), (b), and (c) using abiological sample obtained from said mammal at a subsequent point intime; and (e) comparing the amount of said polynucleotide thathybridizes to said oligonucleotide in step (c) to the amount of saidpolynucleotide that hybridizes to said oligonucleotide in step (d); andtherefrom monitoring the progression of inflammatory bowel disease in amammal. In one embodiment, the oligonucleotide hybridizes undermoderately stringent conditions. In one particular embodiment, thepolymerase chain reaction is used to determine the amount ofpolynucleotide that hybridizes to said oligonucleotide. In anotherembodiment, the amount of polynucleotide that hybridizes to saidoligonucleotide is determined using a hybridization assay. Illustrativebiological samples are sera, stool, tissue or other material obtained bycolonoscopy or colonic biopsy, ileoscopy, esophagogastroduodenoscopy(EGP), or surgery. In a related embodiment, the polynucleotide comprisesa polynucleotide that encodes a flagellin protein.

[0049] Other aspects of the present invention provides diagnostic kitscomprising at least one oligonucleotide as described herein. In relatedaspects, a diagnostic kit may comprise at least one antibody asdescribed herein, and a detection reagent, wherein the detection reagentcomprises a reporter group.

[0050] In certain aspects, the present invention provides a diagnostickit comprising a portion of at least one or more polypeptides describedherein, wherein said portion can be bound by an antibody; and adetection reagent comprising a reporter group. In a related embodiment,said portion of at least one or more polypeptides is immobilized on asolid support. Illustrative detection reagents comprise ananti-immunoglobulin, protein G, protein A, or a lectin. Illustrativereporter groups comprise radioactive groups, fluorescent groups,luminescent groups, enzymes, biotin, or dyes.

[0051] Within yet another aspect, the present invention provides amethod for identifying an inflammatory bowel disease type in a patient,comprising: (a) obtaining an antibody comprising biological sample froma patient; (b) contacting the biological sample with a polypeptide ofclaim 2; (c) detecting in the sample an amount of antibody that binds tothe polypeptide; and (d) comparing the amount of bound antibody to apredetermined value associated with and therefrom subdividing theinflammatory bowel disease type. Within one embodiment the disease typeis selected from ulcerative colitis and Crohn's Disease.

[0052] Within another embodiment the polypeptide comprises a flagellinpolypeptide.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

[0053] SEQ ID NO:1 is the determined cDNA sequence for 76779.

[0054] SEQ ID NO:2 is the determined cDNA sequence for 76780.

[0055] SEQ ID NO:3 is the determined cDNA sequence for 76959.

[0056] SEQ ID NO:4 is the determined cDNA sequence for 76960.

[0057] SEQ ID NO:5 is the determined cDNA sequence for 76961.

[0058] SEQ ID NO:6 is the determined cDNA sequence for 76781.

[0059] SEQ ID NO:7 is the determined cDNA sequence for 76962.

[0060] SEQ ID NO:8 is the determined cDNA sequence for 76782.

[0061] SEQ ID NO:9 is the determined cDNA sequence for 76963.

[0062] SEQ ID NO:10 is the determined cDNA sequence for 76964.

[0063] SEQ ID NO:11 is the determined cDNA sequence for 77529.

[0064] SEQ ID NO:12 is the determined cDNA sequence for 76965.

[0065] SEQ ID NO:13 is the determined cDNA sequence for 76966.

[0066] SEQ ID NO:14 is the determined cDNA sequence for 76967.

[0067] SEQ ID NO:15 is the determined cDNA sequence for 76968.

[0068] SEQ ID NO:16 is the determined cDNA sequence for 77530.

[0069] SEQ ID NO:17 is the determined cDNA sequence for 76969.

[0070] SEQ ID NO:18 is the determined cDNA sequence for 76970.

[0071] SEQ ID NO:19 is the determined cDNA sequence for 76971.

[0072] SEQ ID NO:20 is the determined cDNA sequence for 77073.

[0073] SEQ ID NO:21 is the determined cDNA sequence for 76972.

[0074] SEQ ID NO:22 is the determined cDNA sequence for 76973.

[0075] SEQ ID NO:23 is the determined cDNA sequence for 76974.

[0076] SEQ ID NO:24 is the determined cDNA sequence for 77074.

[0077] SEQ ID NO:25 is the determined cDNA sequence for 77531.

[0078] SEQ ID NO:26 is the determined cDNA sequence for 76975.

[0079] SEQ ID NO:27 is the determined cDNA sequence for 77075.

[0080] SEQ ID NO:28 is the determined cDNA sequence for 76976.

[0081] SEQ ID NO:29 is the determined cDNA sequence for 76977.

[0082] SEQ ID NO:30 is the determined cDNA sequence for 77532.

[0083] SEQ ID NO:31 is the determined cDNA sequence for 77533.

[0084] SEQ ID NO:32 is the determined cDNA sequence for 77534.

[0085] SEQ ID NO:33 is the determined cDNA sequence for 77535.

[0086] SEQ ID NO:34 is the determined cDNA sequence for 77076.

[0087] SEQ ID NO:35 is the determined cDNA sequence for 77536.

[0088] SEQ ID NO:36 is the determined cDNA sequence for 77538.

[0089] SEQ ID NO:37 is the determined cDNA sequence for 77539.

[0090] SEQ ID NO:38 is the amino acid sequence encoded by 76779.

[0091] SEQ ID NO:39 is the amino acid sequence encoded by 76780.

[0092] SEQ ID NO:40 is the amino acid sequence encoded by 76959.

[0093] SEQ ID NO:41 is the amino acid sequence encoded by 76959.

[0094] SEQ ID NO:42 is the amino acid sequence encoded by 76781.

[0095] SEQ ID NO:43 is the amino acid sequence encoded by 76782.

[0096] SEQ ID NO:44 is the amino acid sequence encoded by 76967.

[0097] SEQ ID NO:45 is the amino acid sequence encoded by 76969.

[0098] SEQ ID NO:46 is the amino acid sequence encoded by 76972.

[0099] SEQ ID NO:47 is the amino acid sequence encoded by 76974.

[0100] SEQ ID NO:48 is the amino acid sequence encoded by 76975.

[0101] SEQ ID NO:49 is the amino acid sequence encoded by 76977.

[0102] SEQ ID NO:50 is the amino acid sequence encoded by 77076.

[0103] SEQ ID NO:51 is the determined cDNA sequence for 73261.

[0104] SEQ ID NO:52 is the determined cDNA sequence for 73262.

[0105] SEQ ID NO:53 is the determined cDNA sequence for 73263.

[0106] SEQ ID NO:54 is the determined cDNA sequence for 73264.

[0107] SEQ ID NO:55 is the determined cDNA sequence for 73266.

[0108] SEQ ID NO:56 is the determined cDNA sequence for 73267.

[0109] SEQ ID NO:57 is the determined cDNA sequence for 73268.

[0110] SEQ ID NO:58 is the determined cDNA sequence for 73269.

[0111] SEQ ID NO:59 is the determined cDNA sequence for 73270.

[0112] SEQ ID NO:60 is the determined cDNA sequence for 73272.

[0113] SEQ ID NO:61 is the determined cDNA sequence for 73273.

[0114] SEQ ID NO:62 is the determined cDNA sequence for 73274.

[0115] SEQ ID NO:63 is the determined cDNA sequence for 73275.

[0116] SEQ ID NO:64 is the determined cDNA sequence for 73037.

[0117] SEQ ID NO:65 is the determined cDNA sequence for 75038.

[0118] SEQ ID NO:66 is the determined cDNA sequence for 75039.

[0119] SEQ ID NO:67 is the determined cDNA sequence for 75040.

[0120] SEQ ID NO:68 is the determined cDNA sequence for 75041.

[0121] SEQ ID NO:69 is the determined cDNA sequence for 75042.

[0122] SEQ ID NO:70 is the determined cDNA sequence for 75044.

[0123] SEQ ID NO:71 is the determined cDNA sequence for 75045.

[0124] SEQ ID NO:72 is the determined cDNA sequence for 75046.

[0125] SEQ ID NO:73 is the determined cDNA sequence for 75047.

[0126] SEQ ID NO:74 is the determined cDNA sequence for 75048.

[0127] SEQ ID NO:75 is the full-length determined cDNA sequence for83537, also referred to as Flagellin X.

[0128] SEQ ID NO:76 is the determined cDNA sequence for the aminoterminal conserved end of Flagellin X.

[0129] SEQ ID NO:77 is the determined cDNA sequence for the aminoterminal conserved end plus the variable region of Flagellin X.

[0130] SEQ ID NO:78 is the determined cDNA sequence for thecarboxy-terminal conserved end of Flagellin X.

[0131] SEQ ID NO:79 is the full-length amino acid sequence of FlagellinX.

[0132] SEQ ID NO:80 is the amino acid sequence of the amino terminalconserved end of Flagellin X.

[0133] SEQ ID NO:81 is the amino acid sequence of the amino terminalconserved end plus the variable region of Flagellin X.

[0134] SEQ ID NO:82 is the amino acid sequence of the carboxy-terminalconserved end of Flagellin X.

[0135] SEQ ID NO:83 is the full-length coding sequence of Helicobacterbilis flagellin B.

[0136] SEQ ID NO:84 is the full-length protein sequence of Helicobacterbilis flagellin B, encoded by the nucleotide sequence set forth in SEQID NO:83.

[0137] SEQ ID NO:85 is the full-length coding sequence of Cbir-1flagellin (partial sequence set forth in SEQ ID NO:1).

[0138] SEQ ID NO:86 is the full-length protein sequence of Cbir-1flagellin, encoded by the nucleotide sequence set forth in SEQ ID NO:85.

[0139] SEQ ID NO:87 is the determined full length cDNA sequence forclone 76963 (SEQ ID NO:9), CBir-11.

[0140] SEQ ID NO:88 is a predicted translated protein sequence encodedby SEQ ID NO:86, a flagellin-like sequence.

[0141] SEQ ID NO:89 is a predicted translated protein sequence encodedby SEQ ID NO:86, a phosphoesterase-like sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0142] The present invention is directed generally to compositions andtheir use in the therapy and diagnosis of IBD. As described furtherbelow, illustrative compositions of the present invention include, butare not restricted to, polypeptides, particularly immunogenicpolypeptides, polynucleotides encoding such polypeptides, antibodies andother binding agents, antigen presenting cells (APCs) and immune systemcells (e.g., T and B cells).

[0143] The practice of the present invention will employ, unlessindicated specifically to the contrary, conventional methods ofvirology, immunology, microbiology, molecular biology and recombinantDNA techniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

[0144] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontent clearly dictates otherwise. All references cited herein are eachincorporated by reference in their entirety.

[0145] Polypeptide Compositions

[0146] As used herein, the term “polypeptide” “is used in itsconventional meaning, i.e., as a sequence of amino acids. Thepolypeptides are not limited to a specific length of the product; thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

[0147] Particularly illustrative polypeptides of the present inventioncomprise those encoded by a polynucleotide sequence set forth in any oneof SEQ ID NOs:1-37, 51-78, 83, 85 and 87, or a sequence that hybridizesunder moderately stringent conditions, or, alternatively, under highlystringent conditions, to a polynucleotide sequence set forth in any oneof SEQ ID NOs:1-37, 51-78, 83, 85 and 87. Certain other illustrativepolypeptides of the invention comprise amino acid sequences as set forthin any one of SEQ ID NOs:38-50, 79-82, 84, and 86.

[0148] The polypeptides of the present invention are sometimes hereinreferred to as bacterial proteins or bacterial polypeptides, as anindication that their identification has been based at least in partupon their expression in enteric bacterial samples isolated from thecolon of individuals with IBD. The peptides described herein may beidentified from a lesion in the colon from a patient with IBD.Accordingly, such a peptide may not be present in adjacent normaltissue. Alternatively, a peptide of the present invention may beidentified from an enteric bacterial sample isolated from the colon ofan individual with IBD said enteric bacteria being absent fromindividuals not affected with IBD. In a further embodiment, thepolypeptides of the present invention may be identified by their abilityto activate T cells from individuals affected with IBD. Additionally,polypeptides described herein may be identified by their reactivity withsera from IBD patients as compared to their lack of reactivity to serafrom unaffected individuals.

[0149] Thus, a “bacterial polypeptide” or “bacterial protein,” refersgenerally to a polypeptide sequence of the present invention, or apolynucleotide sequence encoding such a polypeptide, that is present insamples isolated from a substantial proportion of IBD patients, forexample preferably greater than about 20%, more preferably greater thanabout 30%, and most preferably greater than about 50% or more ofpatients tested as determined using a representative assay providedherein. A bacterial polypeptide sequence of the invention, based uponits expression in enteric bacterial samples isolated from the colon ofindividuals with IBD, has particular utility both as a diagnostic markeras well as a therapeutic target, as further described below. In oneparticular embodiment of the present invention, a bacterial polypeptideor bacterial protein comprises a flagellin protein.

[0150] In certain preferred embodiments, the polypeptides of theinvention are immunogenic, i.e., they react detectably within animmunoassay (such as an ELISA or T-cell stimulation assay) with antiseraand/or T-cells from a patient with IBD. Screening for immunogenicactivity can be performed using techniques well known to the skilledartisan. For example, such screens can be performed using methods suchas those described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In one illustrative example, apolypeptide may be immobilized on a solid support and contacted withpatient sera to allow binding of antibodies within the sera to theimmobilized polypeptide. Unbound sera may then be removed and boundantibodies detected using, for example, ¹²⁵I-labeled Protein A.

[0151] As would be recognized by the skilled artisan, immunogenicportions of the polypeptides disclosed herein are also encompassed bythe present invention. An “immunogenic portion,” as used herein, is afragment of an immunogenic polypeptide of the invention that itself isimmunologically reactive (i.e., specifically binds) with the B-cellsand/or T-cell surface antigen receptors that recognize the polypeptide.Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

[0152] In one preferred embodiment, an immunogenic portion of apolypeptide of the present invention is a portion that reacts withantisera and/or T-cells at a level that is not substantially less thanthe reactivity of the full-length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Preferably, the level of immunogenic activityof the immunogenic portion is at least about 50%, preferably at leastabout 70% and most preferably greater than about 90% of theimmunogenicity for the full-length polypeptide. In some instances,preferred immunogenic portions will be identified that have a level ofimmunogenic activity greater than that of the corresponding full-lengthpolypeptide, e.g., having greater than about 100% or 150% or moreimmunogenic activity.

[0153] In certain other embodiments, illustrative immunogenic portionsmay include peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

[0154] In another embodiment, a polypeptide composition of the inventionmay also comprise one or more polypeptides that are immunologicallyreactive with T cells and/or antibodies generated against a polypeptideof the invention, particularly a polypeptide having an amino acidsequence disclosed herein, or to an immunogenic fragment or variantthereof.

[0155] In another embodiment of the invention, polypeptides are providedthat comprise one or more polypeptides that are capable of eliciting Tcells and/or antibodies that are immunologically reactive with one ormore polypeptides described herein, or one or more polypeptides encodedby contiguous nucleic acid sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

[0156] The present invention, in another aspect, provides polypeptidefragments comprising at least about 5, 10, 15, 20, 25, 50, or 100contiguous amino acids, or more, including all intermediate lengths, ofa polypeptide compositions set forth herein, such as those set forth inSEQ ID NOs:38-50, 79-82, 84, 86 and 88-89, or those encoded by apolynucleotide sequence set forth in a sequence of SEQ ID NOs:1-37,51-78, 83, 85 and 87.

[0157] In another aspect, the present invention provides variants of thepolypeptide compositions described herein. Polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity (determined as described below), along itslength, to a polypeptide sequences set forth herein.

[0158] In one preferred embodiment, the polypeptide fragments andvariants provided by the present invention are immunologically reactivewith an antibody and/or T-cell that reacts with a full-lengthpolypeptide specifically set forth herein.

[0159] In another preferred embodiment, the polypeptide fragments andvariants provided by the present invention exhibit a level ofimmunogenic activity of at least about 50%, preferably at least about70%, and most preferably at least about 90% or more of that exhibited bya full-length polypeptide sequence specifically set forth herein.

[0160] A polypeptide “variant,” as the term is used herein, is apolypeptide that typically differs from a polypeptide specificallydisclosed herein in one or more substitutions, deletions, additionsand/or insertions. Such variants may be naturally occurring or may besynthetically generated, for example, by modifying one or more of theabove polypeptide sequences of the invention and evaluating theirimmunogenic activity as described herein and/or using any of a number oftechniques well known in the art.

[0161] For example, certain illustrative variants of the polypeptides ofthe invention include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other illustrative variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

[0162] In many instances, a variant will contain conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. As described above, modifications may be madein the structure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

[0163] For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. It is thus contemplated thatvarious changes may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity. TABLE1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGCUGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

[0164] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0165] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e. still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 (specifically incorporated herein by reference in itsentirety), states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein.

[0166] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4). It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologicallyequivalent protein. In such changes, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those within ±1are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

[0167] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

[0168] In addition, any polynucleotide may be further modified toincrease stability in vivo. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ ends;the use of phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl-, methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

[0169] Amino acid substitutions may further be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

[0170] As noted above, polypeptides may comprise a signal (or leader)sequence at the N-terminal end of the protein, which co-translationallyor post-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

[0171] When comparing polypeptide sequences, two sequences are said tobe “identical” if the sequence of amino acids in the two sequences isthe same when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

[0172] Optimal alignment of sequences for comparison may be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Saitou, N. Nei, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

[0173] Alternatively, optimal alignment of sequences for comparison maybe conducted by the local identity algorithm of Smith and Waterman(1981) Add. APL. Math 2:482, by the identity alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.USA 85: 2444, by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

[0174] One preferred example of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.(1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides and polypeptides of theinvention. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. For aminoacid sequences, a scoring matrix can be used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

[0175] In one preferred approach, the “percentage of sequence identity”is determined by comparing two optimally aligned sequences over a windowof comparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

[0176] Within other illustrative embodiments, a polypeptide may be afusion polypeptide that comprises multiple polypeptides as describedherein, or that comprises at least one polypeptide as described hereinand an unrelated sequence, such as a known bacterial protein. A fusionpartner may, for example, assist in providing T helper epitopes (animmunological fusion partner), preferably T helper epitopes recognizedby humans, or may assist in expressing the protein (an expressionenhancer) at higher yields than the native recombinant protein. Certainpreferred fusion partners are both immunological and expressionenhancing fusion partners. Other fusion partners may be selected so asto increase the solubility of the polypeptide or to enable thepolypeptide to be targeted to desired intracellular compartments. Stillfurther fusion partners include affinity tags, which facilitatepurification of the polypeptide.

[0177] Fusion polypeptides may generally be prepared using standardtechniques, including chemical conjugation. Preferably, a fusionpolypeptide is expressed as a recombinant polypeptide, allowing theproduction of increased levels, relative to a non-fused polypeptide, inan expression system. Briefly, DNA sequences encoding the polypeptidecomponents may be assembled separately, and ligated into an appropriateexpression vector. The 3′ end of the DNA sequence encoding onepolypeptide component is ligated, with or without a peptide linker, tothe 5′ end of a DNA sequence encoding the second polypeptide componentso that the reading frames of the sequences are in phase. This permitstranslation into a single fusion polypeptide that retains the biologicalactivity of both component polypeptides.

[0178] A peptide linker sequence may be employed to separate the firstand second polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiary structures.Such a peptide linker sequence is incorporated into the fusionpolypeptide using standard techniques well known in the art. Suitablepeptide linker sequences may be chosen based on the following factors:(1) their ability to adopt a flexible extended conformation; (2) theirinability to adopt a secondary structure that could interact withfunctional epitopes on the first and second polypeptides; and (3) thelack of hydrophobic or charged residues that might react with thepolypeptide functional epitopes. Preferred peptide linker sequencescontain Gly, Asn and Ser residues. Other near neutral amino acids, suchas Thr and Ala may also be used in the linker sequence. Amino acidsequences which may be usefully employed as linkers include thosedisclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc.Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 andU.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 toabout 50 amino acids in length. Linker sequences are not required whenthe first and second polypeptides have non-essential N-terminal aminoacid regions that can be used to separate the functional domains andprevent steric interference.

[0179] The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

[0180] The fusion polypeptide can comprise a polypeptide as describedherein together with an unrelated immunogenic protein, such as animmunogenic protein capable of eliciting a recall response. Examples ofsuch proteins include tetanus, tuberculosis and hepatitis proteins (see,for example, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

[0181] In one preferred embodiment, the immunological fusion partner isderived from a Mycobacterium sp., such as a Mycobacteriumtuberculosis-derived Ra12 fragment. Ra12 compositions and methods fortheir use in enhancing the expression and/or immunogenicity ofheterologous polynucleotide/polypeptide sequences is described in U.S.Patent Application No. 60/158,585, the disclosure of which isincorporated herein by reference in its entirety. Briefly, Ra12 refersto a polynucleotide region that is a subsequence of a Mycobacteriumtuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 KDmolecular weight encoded by a gene in virulent and avirulent strains ofM. tuberculosis. The nucleotide sequence and amino acid sequence ofMTB32A have been described (for example, U.S. Patent Application No.60/158,585; see also, Skeiky et al., Infection and Immun. (1999)67:3998-4007, incorporated herein by reference). C-terminal fragments ofthe MTB32A coding sequence express at high levels and remain as asoluble polypeptides throughout the purification process. Moreover, Ra12may enhance the immunogenicity of heterologous immunogenic polypeptideswith which it is fused. One preferred Ra12 fusion polypeptide comprisesa 14 KD C-terminal fragment corresponding to amino acid residues 192 to323 of MTB32A. Other preferred Ra12 polynucleotides generally compriseat least about 15 consecutive nucleotides, at least about 30nucleotides, at least about 60 nucleotides, at least about 100nucleotides, at least about 200 nucleotides, or at least about 300nucleotides that encode a portion of a Ra12 polypeptide. Ra12polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Ra12 polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variants preferablyexhibit at least about 70% identity, more preferably at least about 80%identity and most preferably at least about 90% identity to apolynucleotide sequence that encodes a native Ra12 polypeptide or aportion thereof.

[0182] Within other preferred embodiments, an immunological fusionpartner is derived from protein D, a surface protein of thegram-negative bacterium Haemophilus influenza B (WO 91/18926).Preferably, a protein D derivative comprises approximately the firstthird of the protein (e.g., the first N-terminal 100-110 amino acids),and a protein D derivative may be lipidated. Within certain preferredembodiments, the first 109 residues of a Lipoprotein D fusion partner isincluded on the N-terminus to provide the polypeptide with additionalexogenous T-cell epitopes and to increase the expression level in E.coli (thus functioning as an expression enhancer). The lipid tailensures optimal presentation of the antigen to antigen presenting cells.Other fusion partners include the non-structural protein from influenzaevirus, NSI (hemaglutinin). Typically, the N-terminal 81 amino acids areused, although different fragments that include T-helper epitopes may beused.

[0183] In another embodiment, the immunological fusion partner is theprotein known as LYTA, or a portion thereof (preferably a C-terminalportion). LYTA is derived from Streptococcus pneumoniae, whichsynthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encodedby the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin thatspecifically degrades certain bonds in the peptidoglycan backbone. TheC-terminal domain of the LYTA protein is responsible for the affinity tothe choline or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

[0184] Yet another illustrative embodiment involves fusion polypeptides,and the polynucleotides encoding them, wherein the fusion partnercomprises a targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation of CD4⁺T-cells specific for the polypeptide.

[0185] Polypeptides of the invention are prepared using any of a varietyof well known synthetic and/or recombinant techniques, the latter ofwhich are further described below. Polypeptides, portions and othervariants generally less than about 150 amino acids can be generated bysynthetic means, using techniques well known to those of ordinary skillin the art. In one illustrative example, such polypeptides aresynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions.

[0186] In general, polypeptide compositions (including fusionpolypeptides) of the invention are isolated. An “isolated” polypeptideis one that is removed from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

[0187] Polynucleotide Compositions

[0188] The present invention, in other aspects, provides polynucleotidecompositions. The terms “DNA” and “polynucleotide” are used essentiallyinterchangeably herein to refer to a DNA molecule that has been isolatedfree of total genomic DNA of a particular species. “Isolated,” as usedherein, means that a polynucleotide is substantially away from othercoding sequences, and that the DNA molecule does not contain largeportions of unrelated coding DNA, such as large chromosomal fragments orother functional genes or polypeptide coding regions. Of course, thisrefers to the DNA molecule as originally isolated, and does not excludegenes or coding regions later added to the segment by the hand of man.

[0189] As will be understood by those skilled in the art, thepolynucleotide compositions of this invention can include genomicsequences, extra-genomic and plasmid-encoded sequences and smallerengineered gene segments that express, or may be adapted to express,proteins, polypeptides, peptides and the like. Such segments may benaturally isolated, or modified synthetically by the hand of man.

[0190] As will be also recognized by the skilled artisan,polynucleotides of the invention may be single-stranded (coding orantisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules may include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or noncoding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

[0191] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes a polypeptide/protein of the inventionor a portion thereof) or may comprise a sequence that encodes a variantor derivative, preferably and immunogenic variant or derivative, of sucha sequence.

[0192] Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that comprise some or all of apolynucleotide sequence set forth in any one of SEQ ID NOs:1-37, 51-78,83, 85 and 87, complements of a polynucleotide sequence set forth in anyone of SEQ ID NOs:1-37, 51-78, 83, 85 and 87, and degenerate variants ofa polynucleotide sequence set forth in any one of SEQ ID NOs:1-37,51-78, 83, 85 and 87. In certain preferred embodiments, thepolynucleotide sequences set forth herein encode immunogenicpolypeptides, as described above. In other certain preferredembodiments, the polynucloetide sequences set forth herein encodeIBD-associated bacterial proteins isolated as described herein fromindividuals affected with IBD. In other certain preferred embodiments,the polynucloetide sequences set forth herein encode flagellin proteins.

[0193] In other related embodiments, the present invention providespolynucleotide variants having substantial identity to the sequencesdisclosed herein in SEQ ID NOs:1-37, 51-78, 83, 85, and 87, for examplethose comprising at least 70% sequence identity, preferably at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequenceidentity compared to a polynucleotide sequence of this invention usingthe methods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

[0194] Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompasses homologousgenes of xenogenic origin.

[0195] In additional embodiments, the present invention providespolynucleotide fragments comprising or consisting of various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise or consist of at least about10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or morecontiguous nucleotides of one or more of the sequences disclosed hereinas well as all intermediate lengths there between. It will be readilyunderstood that “intermediate lengths”, in this context, means anylength between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22,23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103,etc.; 150, 151, 152, 153, etc.; including all integers through 200-500;500-1,000, and the like. A polynucleotide sequence as described here maybe extended at one or both ends by additional nucleotides not found inthe native sequence. This additional sequence may consist of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotidesat either end of the disclosed sequence or at both ends of the disclosedsequence.

[0196] In another embodiment of the invention, polynucleotidecompositions are provided that are capable of hybridizing under moderateto high stringency conditions to a polynucleotide sequence providedherein, or a fragment thereof, or a complementary sequence thereof.Hybridization techniques are well known in the art of molecular biology.For purposes of illustration, suitable moderately stringent conditionsfor testing the hybridization of a polynucleotide of this invention withother polynucleotides include prewashing in a solution of 5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC,overnight; followed by washing twice at 65° C. for 20 minutes with eachof 2×, 0.5× and 0.2×SSC containing 0.1% SDS. One skilled in the art willunderstand that the stringency of hybridization can be readilymanipulated, such as by altering the salt content of the hybridizationsolution and/or the temperature at which the hybridization is performed.For example, in another embodiment, suitable highly stringenthybridization conditions include those described above, with theexception that the temperature of hybridization is increased, e.g., to60-65° C. or 65-70° C.

[0197] In certain preferred embodiments, the polynucleotides describedabove, e.g., polynucleotide variants, fragments and hybridizingsequences, encode polypeptides that are immunologically cross-reactivewith a polypeptide sequence specifically set forth herein. In otherpreferred embodiments, such polynucleotides encode polypeptides thathave a level of immunogenic activity of at least about 50%, preferablyat least about 70%, and more preferably at least about 90% of that for apolypeptide sequence specifically set forth herein.

[0198] The polynucleotides of the present invention, or fragmentsthereof, regardless of the length of the coding sequence itself, may becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol. For example, illustrativepolynucleotide segments with total lengths of about 10,000, about 5000,about 3000, about 2,000, about 1,000, about 500, about 200, about 100,about 50 base pairs in length, and the like, (including all intermediatelengths) are contemplated to be useful in many implementations of thisinvention.

[0199] When comparing polynucleotide sequences, two sequences are saidto be “identical” if the sequence of nucleotides in the two sequences isthe same when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

[0200] Optimal alignment of sequences for comparison may be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

[0201] Alternatively, optimal alignment of sequences for comparison maybe conducted by the local identity algorithm of Smith and Waterman(1981) Add. APL. Math 2:482, by the identity alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.USA 85: 2444, by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

[0202] One preferred example of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.(1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparisonof both strands.

[0203] Preferably, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

[0204] It will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention. Further, alleles of the genes comprising thepolynucleotide sequences provided herein are within the scope of thepresent invention. Alleles are endogenous genes that are altered as aresult of one or more mutations, such as deletions, additions and/orsubstitutions of nucleotides. The resulting mRNA and protein may, butneed not, have an altered structure or function. Alleles may beidentified using standard techniques (such as hybridization,amplification and/or database sequence comparison).

[0205] Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of immunogenic variants and/or derivatives of thepolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

[0206] Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

[0207] In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

[0208] As will be appreciated by those of skill in the art,site-specific mutagenesis techniques have often employed a phage vectorthat exists in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage. These phage are readily commercially-available and their useis generally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

[0209] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

[0210] The preparation of sequence variants of the selectedpeptide-encoding DNA segments using site-directed mutagenesis provides ameans of producing potentially useful species and is not meant to belimiting as there are other ways in which sequence variants of peptidesand the DNA sequences encoding them may be obtained. For example,recombinant vectors encoding the desired peptide sequence may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants. Specific details regarding these methods and protocols arefound in the teachings of Maloy et al., 1994; Segal, 1976; Prokop andBajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporatedherein by reference, for that purpose.

[0211] As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

[0212] In another approach for the production of polypeptide variants ofthe present invention, recursive sequence recombination, as described inU.S. Pat. No. 5,837,458, may be employed. In this approach, iterativecycles of recombination and screening or selection are performed to“evolve” individual polynucleotide variants of the invention having, forexample, enhanced immunogenic activity.

[0213] In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise or consist of a sequence region ofat least about a 15 nucleotide long contiguous sequence that has thesame sequence as, or is complementary to, a 15 nucleotide longcontiguous sequence disclosed herein will find particular utility.Longer contiguous identical or complementary sequences, e.g., those ofabout 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediatelengths) and even up to full length sequences will also be of use incertain embodiments.

[0214] The ability of such nucleic acid probes to specifically hybridizeto a sequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

[0215] Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

[0216] The use of a hybridization probe of about 15-25 nucleotides inlength allows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

[0217] Hybridization probes may be selected from any portion of any ofthe sequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences may be governed byvarious factors. For example, one may wish to employ primers fromtowards the termini of the total sequence.

[0218] Small polynucleotide segments or fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, as is commonly practiced using an automated oligonucleotidesynthesizer. Also, fragments may be obtained by application of nucleicacid reproduction technology, such as the PCR™ technology of U.S. Pat.No. 4,683,202, by introducing selected sequences into recombinantvectors for recombinant production, and by other recombinant DNAtechniques generally known to those of skill in the art of molecularbiology.

[0219] The nucleotide sequences of the invention may be used for theirability to selectively form duplex molecules with complementarystretches of the entire gene or gene fragments of interest. Depending onthe application envisioned, one will typically desire to employ varyingconditions of hybridization to achieve varying degrees of selectivity ofprobe towards target sequence. For applications requiring highselectivity, one will typically desire to employ relatively stringentconditions to form the hybrids, e.g., one will select relatively lowsalt and/or high temperature conditions, such as provided by a saltconcentration of from about 0.02 M to about 0.15 M salt at temperaturesof from about 50° C. to about 70° C. Such selective conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand, and would be particularly suitable for isolating relatedsequences.

[0220] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0221] According to another embodiment of the present invention,polynucleotide compositions comprising antisense oligonucleotides areprovided. Antisense oligonucleotides have been demonstrated to beeffective and targeted inhibitors of protein synthesis, and,consequently, provide a therapeutic approach by which a disease can betreated by inhibiting the synthesis of proteins that contribute to thedisease. The efficacy of antisense oligonucleotides for inhibitingprotein synthesis is well established. For example, the synthesis ofpolygalactauronase and the muscarine type 2 acetylcholine receptor areinhibited by antisense oligonucleotides directed to their respectivemRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829).Further, examples of antisense inhibition have been demonstrated withthe nuclear protein cyclin, the multiple drug resistance gene (MDG1),ICAM-1, E-selectin, STK-1, striatal GABAA receptor and human EGF(Jaskulski et al., Science. Jun. 10, 1988; 240(4858):1544-6;Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al.,Brain Res Mol Brain Res. Jun. 15, 1998; 57(2):310-20; U.S. Pat. No.5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S.Pat. No. 5,610,288). Antisense constructs have also been described thatinhibit and can be used to treat a variety of abnormal cellularproliferations, e.g. cancer (U.S. Pat. No. 5,747,470; U.S. Pat. No.5,591,317 and U.S. Pat. No. 5,783,683).

[0222] Therefore, in certain embodiments, the present invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein. Selection of antisense compositions specific for a given genesequence is based upon analysis of the chosen target sequence anddetermination of secondary structure, T_(m), binding energy, andrelative stability. Antisense compositions may be selected based upontheir relative inability to form dimers, hairpins, or other secondarystructures that would reduce or prohibit specific binding to the targetmRNA in a host cell. Highly preferred target regions of the mRNA, arethose which are at or near the AUG translation initiation codon, andthose sequences which are substantially complementary to 5′ regions ofthe mRNA. These secondary structure analyses and target site selectionconsiderations can be performed, for example, using v.4 of the OLIGOprimer analysis software and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0223] The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., Nucleic Acids Res. Jul. 15, 1997;25(14):2730-6). It has been demonstrated that several molecules of theMPG peptide coat the antisense oligonucleotides and can be deliveredinto cultured mammalian cells in less than 1 hour with relatively highefficiency (90%). Further, the interaction with MPG strongly increasesboth the stability of the oligonucleotide to nuclease and the ability tocross the plasma membrane.

[0224] According to another embodiment of the invention, thepolynucleotide compositions described herein are used in the design andpreparation of ribozyme molecules for inhibiting expression of thebacterial polypeptides and proteins of the present invention inbacterial cells. Ribozymes are RNA-protein complexes that cleave nucleicacids in a site-specific fashion. Ribozymes have specific catalyticdomains that possess endonuclease activity (Kim and Cech, Proc Natl AcadSci USA. 1987 December; 84(24):8788-92; Forster and Symons, Cell. Apr.24, 1987; 49(2):211-20). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., Cell. 1981 December; 27(3 Pt2):487-96; Michel and Westhof, J Mol. Biol. 1990 December 5;216(3):585-610; Reinhold-Hurek and Shub, Nature. May 14, 1992;357(6374):173-6). This specificity has been attributed to therequirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

[0225] Six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

[0226] The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., Proc Natl Acad SciUSA. Aug. 15, 1992; 89(16):7305-9). Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

[0227] The enzymatic nucleic acid molecule may be formed in ahammerhead, hairpin, a hepatitis δ virus, group I intron or RNaseP RNA(in association with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. NucleicAcids Res. Sep. 11, 1992; 20(17):4559-65. Examples of hairpin motifs aredescribed by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),Hampel and Tritz, Biochemistry Jun. 13, 1989; 28(12):4929-33; Hampel etal., Nucleic Acids Res. Jan. 25, 1990; 18(2):299-304 and U.S. Pat. No.5,631,359. An example of the hepatitis δ virus motif is described byPerrotta and Been, Biochemistry. Dec. 1, 1992; 31(47):11843-52; anexample of the RNaseP motif is described by Guerrier-Takada et al.,Cell. 1983 December; 35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motifis described by Collins (Saville and Collins, Cell. May 18, 1990;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. Oct. 1, 1991;88(19):8826-30; Collins and Olive, Biochemistry. Mar. 23, 1993;32(11):2795-9); and an example of the Group I intron is described in(U.S. Pat. No. 4,987,071). All that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target geneRNA regions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

[0228] Ribozymes may be designed as described in Int. Pat. Appl. Publ.No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595) andsynthesized to be tested in vitro and in vivo, as described. Suchribozymes can also be optimized for delivery. While specific examplesare provided, those in the art will recognize that equivalent RNAtargets in other species can be utilized when necessary.

[0229] Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

[0230] Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describesthe general methods for delivery of enzymatic RNA molecules. Ribozymesmay be administered to cells by a variety of methods known to thosefamiliar to the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569.

[0231] Another means of accumulating high concentrations of aribozyme(s) within cells is to incorporate the ribozyme-encodingsequences into a DNA expression vector. Transcription of the ribozymesequences are driven from a promoter for eukaryotic RNA polymerase I(pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).Transcripts from pol II or pol III promoters will be expressed at highlevels in all cells; the levels of a given pol II promoter in a givencell type will depend on the nature of the gene regulatory sequences(enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerasepromoters may also be used, providing that the prokaryotic RNApolymerase enzyme is expressed in the appropriate cells Ribozymesexpressed from such promoters have been shown to function in mammaliancells. Such transcription units can be incorporated into a variety ofvectors for introduction into mammalian cells, including but notrestricted to, plasmid DNA vectors, viral DNA vectors (such asadenovirus or adeno-associated vectors), or viral RNA vectors (such asretroviral, semliki forest virus, sindbis virus vectors).

[0232] In another embodiment of the invention, peptide nucleic acids(PNAs) compositions are provided. PNA is a DNA mimic in which thenucleobases are attached to a pseudopeptide backbone (Good and Nielsen,Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to beutilized in a number methods that traditionally have used RNA or DNA.Often PNA sequences perform better in techniques than the correspondingRNA or DNA sequences and have utilities that are not inherent to RNA orDNA. A review of PNA including methods of making, characteristics of,and methods of using, is provided by Corey (Trends Biotechnol 1997 June;15(6):224-9). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of the ACE mRNAsequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

[0233] PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science Dec. 6, 1991;254(5037):1497-500; Hanvey et al., Science. Nov. 27, 1992;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 January;4(1):5-23). This chemistry has three important consequences: firstly, incontrast to DNA or phosphorothioate oligonucleotides, PNAs are neutralmolecules; secondly, PNAs are achirial, which avoids the need to developa stereoselective synthesis; and thirdly, PNA synthesis uses standardBoc or Fmoc protocols for solid-phase peptide synthesis, although othermethods, including a modified Merrifield method, have been used.

[0234] PNA monomers or ready-made oligomers are commercially availablefrom PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by eitherBoc or Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg Med Chem. 1995 April; 3(4):437-45). Themanual protocol lends itself to the production of chemically modifiedPNAs or the simultaneous synthesis of families of closely related PNAs.

[0235] As with peptide synthesis, the success of a particular PNAsynthesis will depend on the properties of the chosen sequence. Forexample, while in theory PNAs can incorporate any combination ofnucleotide bases, the presence of adjacent purines can lead to deletionsof one or more residues in the product. In expectation of thisdifficulty, it is suggested that, in producing PNAs with adjacentpurines, one should repeat the coupling of residues likely to be addedinefficiently. This should be followed by the purification of PNAs byreverse-phase high-pressure liquid chromatography, providing yields andpurity of product similar to those observed during the synthesis ofpeptides.

[0236] Modifications of PNAs for a given application may be accomplishedby coupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med Chem. 1995 April; 3(4):437-45; Petersen et al., J PeptSci. 1995 May-June; 1(3):175-83; Orum et al., Biotechniques. 1995September; 19(3):472-80; Footer et al., Biochemistry. Aug. 20, 1996;35(33): 10673-9; Griffith et al., Nucleic Acids Res. Aug. 11, 1995;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci USA. Jun. 6, 1995;92(12):5592-6; Boffa et al., Proc Natl Acad Sci USA. Mar. 14, 1995;92(6):1901-5; Gambacorti-Passerini et al., Blood. Aug. 15, 1996;88(4):1411-7; Armitage et al., Proc Natl Acad Sci USA. Nov. 11, 1997;94(23):12320-5; Seeger et al., Biotechniques. 1997 September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimericmolecules and their uses in diagnostics, modulating protein inorganisms, and treatment of conditions susceptible to therapeutics.

[0237] Methods of characterizing the antisense binding properties ofPNAs are discussed in Rose (Anal Chem. Dec. 15, 1993; 65(24):3545-9) andJensen et al. (Biochemistry. Apr. 22, 1997; 36(16):5072-7). Rose usescapillary gel electrophoresis to determine binding of PNAs to theircomplementary oligonucleotide, measuring the relative binding kineticsand stoichiometry. Similar types of measurements were made by Jensen etal. using BIAcore™ technology.

[0238] Other applications of PNAs that have been described and will beapparent to the skilled artisan include use in DNA strand invasion,antisense inhibition, mutational analysis, enhancers of transcription,nucleic acid purification, isolation of transcriptionally active genes,blocking of transcription factor binding, genome cleavage, biosensors,in situ hybridization, and the like.

[0239] Polynucleotide Identification Characterization and Expression

[0240] Polynucleotides compositions of the present invention may beidentified, prepared and/or manipulated using any of a variety of wellestablished techniques (see generally, Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y., 1989, and other like references). For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for bacterial cDNAs present in tissuesamples isolated from individuals affected with IBD as compared tosamples isolated from unaffected individuals. Such screens may beperformed, for example, using the microarray technology of Affymetrix,Inc. (Santa Clara, Calif.) according to the manufacturer's instructions(and essentially as described by Schena et al., Proc. Natl. Acad. Sci.USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155, 1997). Alternatively, polynucleotide compositions of thepresent invention may be identified by screening mouse or human cecalbacteria genomic random shear expression libraries, as described inExample 1.

[0241] Alternatively, polynucleotides may be amplified from cDNAprepared from cells expressing the bacterial proteins described herein.Many template dependent processes are available to amplify a targetsequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

[0242] Any of a number of other template dependent processes, many ofwhich are variations of the PCR™ amplification technique, are readilyknown and available in the art. Illustratively, some such methodsinclude the ligase chain reaction (referred to as LCR), described, forexample, in Eur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No.4,883,750; Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.PCT/US87/00880; Strand Displacement Amplification (SDA) and Repair ChainReaction (RCR). Still other amplification methods are described in GreatBritain Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025. Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (PCT Intl. Pat. Appl.Publ. No. WO 88/10315), including nucleic acid sequence basedamplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822describes a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods such as “RACE” (Frohman, 1990), and “one-sidedPCR” (Ohara, 1989) are also well-known to those of skill in the art.

[0243] An amplified portion of a polynucleotide of the present inventionmay be used to isolate a full length gene from a suitable library (e.g.,a bacterial cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

[0244] For hybridization techniques, a partial sequence may be labeled(e.g., by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

[0245] Alternatively, amplification techniques, such as those describedabove, can be useful for obtaining a full length coding sequence from apartial cDNA sequence. One such amplification technique is inverse PCR(see Triglia et al., Nucl. Acids Res. 16:8186, 1988), which usesrestriction enzymes to generate a fragment in the known region of thegene. The fragment is then circularized by intramolecular ligation andused as a template for PCR with divergent primers derived from the knownregion. Within an alternative approach, sequences adjacent to a partialsequence may be retrieved by amplification with a primer to a linkersequence and a primer specific to a known region. The amplifiedsequences are typically subjected to a second round of amplificationwith the same linker primer and a second primer specific to the knownregion. A variation on this procedure, which employs two primers thatinitiate extension in opposite directions from the known sequence, isdescribed in WO 96/38591. Another such technique is known as “rapidamplification of cDNA ends” or RACE. This technique involves the use ofan internal primer and an external primer, which hybridizes to a polyAregion or vector sequence, to identify sequences that are 5′ and 3′ of aknown sequence. Additional techniques include capture PCR (Lagerstrom etal., PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al.,Nucl. Acids. Res. 19:3055-60, 1991). Other methods employingamplification may also be employed to obtain a full length cDNAsequence.

[0246] In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

[0247] In other embodiments of the invention, polynucleotide sequencesor fragments thereof which encode polypeptides of the invention, orfusion proteins or functional equivalents thereof, may be used inrecombinant DNA molecules to direct expression of a polypeptide inappropriate host cells. Due to the inherent degeneracy of the geneticcode, other DNA sequences that encode substantially the same or afunctionally equivalent amino acid sequence may be produced and thesesequences may be used to clone and express a given polypeptide.

[0248] As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

[0249] Moreover, the polynucleotide sequences of the present inventioncan be engineered using methods generally known in the art in order toalter polypeptide encoding sequences for a variety of reasons, includingbut not limited to, alterations which modify the cloning, processing,and/or expression of the gene product. For example, DNA shuffling byrandom fragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

[0250] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

[0251] Sequences encoding a desired polypeptide may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of a polypeptide, or a portionthereof. For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).

[0252] A newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.) or other comparable techniques available in theart. The composition of the synthetic peptides may be confirmed by aminoacid analysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

[0253] In order to express a desired polypeptide, the nucleotidesequences encoding the polypeptide, or functional equivalents, may beinserted into appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods which are well known to thoseskilled in the art may be used to construct expression vectorscontaining sequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.

[0254] A variety of expression vector/host systems may be utilized tocontain and express polynucleotide sequences. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0255] The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thepBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

[0256] In bacterial systems, any of a number of expression vectors maybe selected depending upon the use intended for the expressedpolypeptide. For example, when large quantities are needed, for examplefor the induction of antibodies, vectors which direct high levelexpression of fusion proteins that are readily purified may be used.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as pBLUESCRIPT (Stratagene), inwhich the sequence encoding the polypeptide of interest may be ligatedinto the vector in frame with sequences for the amino-terminal Met andthe subsequent 7 residues of .beta.-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0257] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0258] In cases where plant expression vectors are used, the expressionof sequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0259] An insect system may also be used to express a polypeptide ofinterest. For example, in one such system, Autographa californicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.The sequences encoding the polypeptide may be cloned into anon-essential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe polypeptide-encoding sequence will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which the polypeptide ofinterest may be expressed (Engelhard, E. K. et al. (1994) Proc. Natl.Acad. Sci. 91:3224-3227).

[0260] In mammalian host cells, a number of viral-based expressionsystems are generally available. For example, in cases where anadenovirus is used as an expression vector, sequences encoding apolypeptide of interest may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing the polypeptide in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0261] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding a polypeptide of interest.Such signals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

[0262] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0263] For long-term, high-yield production of recombinant proteins,stable expression is generally preferred. For example, cell lines whichstably express a polynucleotide of interest may be transformed usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

[0264] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990)Cell 22:817-23) genes which can be employed in tk.sup.- oraprt.sup.-cells, respectively. Also, antimetabolite, antibiotic orherbicide resistance can be used as the basis for selection; forexample, dhfr which confers resistance to methotrexate (Wigler, M. etal. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confersresistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin,F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which conferresistance to chlorsulfuron and phosphinotricin acetyltransferase,respectively (Murry, supra). Additional selectable genes have beendescribed, for example, trpB, which allows cells to utilize indole inplace of tryptophan, or hisD, which allows cells to utilize histinol inplace of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. 85:8047-51). The use of visible markers has gained popularitywith such markers as anthocyanins, beta-glucuronidase and its substrateGUS, and luciferase and its substrate luciferin, being widely used notonly to identify transformants, but also to quantify the amount oftransient or stable protein expression attributable to a specific vectorsystem (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0265] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0266] Alternatively, host cells that contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include, for example, membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein.

[0267] A variety of protocols for detecting and measuring the expressionof polynucleotide-encoded products, using either polyclonal ormonoclonal antibodies specific for the product are known in the art.Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on a given polypeptide may bepreferred for some applications, but a competitive binding assay mayalso be employed. These and other assays are described, among otherplaces, in Hampton, R. et al. (1990; Serological Methods, a LaboratoryManual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J.Exp. Med. 158:1211-1216).

[0268] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences, or any portionsthereof may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0269] Host cells transformed with a polynucleotide sequence of interestmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides ofthe invention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

[0270] In addition to recombinant production methods, polypeptides ofthe invention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

[0271] Antibody Compositions, Fragments Thereof and Other Binding Agents

[0272] According to another aspect, the present invention furtherprovides binding agents, such as antibodies and antigen-bindingfragments thereof, that exhibit immunological binding to a bacterialpolypeptide disclosed herein, or to a portion, variant or derivativethereof. In one particular embodiment, the antibodies of the presentinvention bind to a Toll-like receptor. Illustrative Toll-like receptors(TLR) include, but are not limited to, TLR5. In a related embodiment,the antibodies of the present invention may bind to a flagellin protein.In another embodiment, the antibodies of the present invention areneutralizing antibodies that block the interaction between TLR5 and aflagellin protein.

[0273] An antibody, or antigen-binding fragment thereof, is said to“specifically bind,” “immunogically bind,” and/or is “immunologicallyreactive” to a polypeptide of the invention if it reacts at a detectablelevel (within, for example, an ELISA assay) with the polypeptide, anddoes not react detectably with unrelated polypeptides under similarconditions.

[0274] Immunological binding, as used in this context, generally refersto the non-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

[0275] An “antigen-binding site,” or “binding portion” of an antibodyrefers to the part of the immunoglobulin molecule that participates inantigen binding. The antigen binding site is formed by amino acidresidues of the N-terminal variable (“V”) regions of the heavy (“H”) andlight (“L”) chains. Three highly divergent stretches within the Vregions of the heavy and light chains are referred to as “hypervariableregions” which are interposed between more conserved flanking stretchesknown as “framework regions,” or “FRs”. Thus the term “FR” refers toamino acid sequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

[0276] Binding agents may be further capable of differentiating betweenpatients with and without IBD, using the representative assays providedherein. For example, antibodies or other binding agents that bind to abacterial protein will preferably generate a signal indicating thepresence of IBD in at least about 20% of patients with the disease, morepreferably at least about 30% of patients. Alternatively, or inaddition, the antibody will generate a negative signal indicating theabsence of the disease in at least about 90% of individuals without IBD.To determine whether a binding agent satisfies this requirement,biological samples (e.g., blood, sera, sputum, urine, feces, and/orbiopsies) from patients with and without IBD (as determined usingstandard clinical tests) may be assayed as described herein for thepresence of polypeptides that bind to the binding agent. Preferably, astatistically significant number of samples with and without the diseasewill be assayed. Each binding agent should satisfy the above criteria;however, those of ordinary skill in the art will recognize that bindingagents may be used in combination to improve sensitivity.

[0277] Binding agents may be further capable of identifying patients atrisk for developing IBD, using the representative assays providedherein. For example, antibodies or other binding agents that bind to abacterial protein will preferably generate a signal indicating a riskfor the development of IBD in at least about 20% of patients withpositive family history of the disease, or patients with a definedgenetic risk for developing the disease (for example, individualspositive for the NOD2 mutation), more preferably at least about 30% ofsaid individuals. Alternatively, or in addition, the antibody willgenerate a negative signal indicating the absence of risk for developingdisease in at least about 90% of individuals with no family history orwith no defined genetic risk of developing IBD. To determine whether abinding agent satisfies this requirement, biological samples (e.g.,blood, sera, sputum, urine, feces, and/or biopsies) from patients withand without risk factors for the development of IBD (as determined usingstandard clinical tests) may be assayed as described herein for thepresence of polypeptides that bind to the binding agent. Preferably, astatistically significant number of samples from individuals with andwithout risk of developing the disease will be assayed. Each bindingagent should satisfy the above criteria; however, those of ordinaryskill in the art will recognize that binding agents may be used incombination to improve sensitivity.

[0278] Binding agents of the present invention may be further used,either alone or in combination with other diagnostic modalities, tosubdivide IBD patients into categories of disease that would besusceptible or resistant to new or existing treatments. In particular,serum reactivity against the binding agents to subdivide IBD patientswould be useful as a stand alone diagnostic or in combination with otherknown diagnostic markers and assays, such as pANCA. Such binding agentscould also be used to distinquish clinical subgroups of patients withCrohn's Disease, which would be of relevance to predict the clinicalcourse of an individual patient or predict responsivensess to particularmedication. Diagnostic use may be as a stand-alone test or used incombination with other serological testing methods or in combinationwith standard laboratory, clinical or pathological testing or withDNA-based testing such as for NOD2 mutations.

[0279] Any agent that satisfies the above requirements may be a bindingagent. For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

[0280] Monoclonal antibodies specific for an antigenic polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

[0281] Monoclonal antibodies may be isolated from the supernatants ofgrowing hybridoma colonies. In addition, various techniques may beemployed to enhance the yield, such as injection of the hybridoma cellline into the peritoneal cavity of a suitable vertebrate host, such as amouse. Monoclonal antibodies may then be harvested from the ascitesfluid or the blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

[0282] A number of therapeutically useful molecules are known in the artwhich comprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0283] A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

[0284] Each of the above-described molecules includes a heavy chain anda light chain CDR set, respectively interposed between a heavy chain anda light chain FR set which provide support to the CDRS and define thespatial relationship of the CDRs relative to each other. As used herein,the term “CDR set” refers to the three hypervariable regions of a heavyor light chain V region. Proceeding from the N-terminus of a heavy orlight chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

[0285] As used herein, the term “FR set” refers to the four flankingamino acid sequences which frame the CDRs of a CDR set of a heavy orlight chain V region. Some FR residues may contact bound antigen;however, FRs are primarily responsible for folding the V region into theantigen-binding site, particularly the FR residues directly adjacent tothe CDRS. Within FRs, certain amino residues and certain structuralfeatures are very highly conserved. In this regard, all V regionsequences contain an internal disulfide loop of around 90 amino acidresidues. When the V regions fold into a binding-site, the CDRs aredisplayed as projecting loop motifs which form an antigen-bindingsurface. It is generally recognized that there are conserved structuralregions of FRs which influence the folded shape of the CDR loops intocertain “canonical” structures—regardless of the precise CDR amino acidsequence. Further, certain FR residues are known to participate innon-covalent interdomain contacts which stabilize the interaction of theantibody heavy and light chains.

[0286] A number of “humanized” antibody molecules comprising anantigen-binding site derived from a non-human immunoglobulin have beendescribed, including chimeric antibodies having rodent V regions andtheir associated CDRs fused to human constant domains (Winter et al.(1991) Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci.USA 86:4220-4224; Shaw et al. (1987) J. Immunol. 138:4534-4538; andBrown et al. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted intoa human supporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

[0287] As used herein, the terms “veneered FRs” and “recombinantlyveneered FRs” refer to the selective replacement of FR residues from,e.g., a rodent heavy or light chain V region, with human FR residues inorder to provide a xenogeneic molecule comprising an antigen-bindingsite which retains substantially all of the native FR polypeptidefolding structure. Veneering techniques are based on the understandingthat the ligand binding characteristics of an antigen-binding site aredetermined primarily by the structure and relative disposition of theheavy and light chain CDR sets within the antigen-binding surface.Davies et al. (1990) Ann. Rev. Biochem. 59:439-473. Thus, antigenbinding specificity can be preserved in a humanized antibody onlywherein the CDR structures, their interaction with each other, and theirinteraction with the rest of the V region domains are carefullymaintained. By using veneering techniques, exterior (e.g.,solvent-accessible) FR residues which are readily encountered by theimmune system are selectively replaced with human residues to provide ahybrid molecule that comprises either a weakly immunogenic, orsubstantially non-immunogenic veneered surface.

[0288] The process of veneering makes use of the available sequence datafor human antibody variable domains compiled by Kabat et al., inSequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. ofHealth and Human Services, U.S. Government Printing Office, 1987),updates to the Kabat database, and other accessible U.S. and foreigndatabases (both nucleic acid and protein). Solvent accessibilities of Vregion amino acids can be deduced from the known three-dimensionalstructure for human and murine antibody fragments. There are two generalsteps in veneering a murine antigen-binding site. Initially, the FRs ofthe variable domains of an antibody molecule of interest are comparedwith corresponding FR sequences of human variable domains obtained fromthe above-identified sources. The most homologous human V regions arethen compared residue by residue to corresponding murine amino acids.The residues in the murine FR which differ from the human counterpartare replaced by the residues present in the human moiety usingrecombinant techniques well known in the art. Residue switching is onlycarried out with moieties which are at least partially exposed (solventaccessible), and care is exercised in the replacement of amino acidresidues which may have a significant effect on the tertiary structureof V region domains, such as proline, glycine and charged amino acids.

[0289] In this manner, the resultant “veneered” murine antigen-bindingsites are thus designed to retain the murine CDR residues, the residuessubstantially adjacent to the CDRs, the residues identified as buried ormostly buried (solvent inaccessible), the residues believed toparticipate in non-covalent (e.g., electrostatic and hydrophobic)contacts between heavy and light chain domains, and the residues fromconserved structural regions of the FRs which are believed to influencethe “canonical” tertiary structures of the CDR loops. These designcriteria are then used to prepare recombinant nucleotide sequences whichcombine the CDRs of both the heavy and light chain of a murineantigen-binding site into human-appearing FRs that can be used totransfect mammalian cells for the expression of recombinant humanantibodies which exhibit the antigen specificity of the murine antibodymolecule.

[0290] In another embodiment of the invention, monoclonal antibodies ofthe present invention may be coupled to one or more therapeutic agents.Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

[0291] A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

[0292] Alternatively, it may be desirable to couple a therapeutic agentand an antibody via a linker group. A linker group can function as aspacer to distance an antibody from an agent in order to avoidinterference with binding capabilities. A linker group can also serve toincrease the chemical reactivity of a substituent on an agent or anantibody, and thus increase the coupling efficiency. An increase inchemical reactivity may also facilitate the use of agents, or functionalgroups on agents, which otherwise would not be possible.

[0293] It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

[0294] Where a therapeutic agent is more potent when free from theantibody portion of the immunoconjugates of the present invention, itmay be desirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

[0295] It may be desirable to couple more than one agent to an antibody.In one embodiment, multiple molecules of an agent are coupled to oneantibody molecule. In another embodiment, more than one type of agentmay be coupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

[0296] A carrier may bear the agents in a variety of ways, includingcovalent bonding either directly or via a linker group. Suitablecarriers include proteins such as albumins (e.g., U.S. Pat. No.4,507,234, to Kato et al.), peptides and polysaccharides such asaminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carriermay also bear an agent by noncovalent bonding or by encapsulation, suchas within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and4,873,088). Carriers specific for radionuclide agents includeradiohalogenated small molecules and chelating compounds. For example,U.S. Pat. No. 4,735,792 discloses representative radiohalogenated smallmolecules and their synthesis. A radionuclide chelate may be formed fromchelating compounds that include those containing nitrogen and sulfuratoms as the donor atoms for binding the metal, or metal oxide,radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et al.discloses representative chelating compounds and their synthesis.

[0297] T Cell Compositions

[0298] The present invention, in another aspect, provides T cellsspecific for a bacterial polypeptide disclosed herein, or for a variantor derivative thereof. Such cells may generally be prepared in vitro orex vivo, using standard procedures. For example, T cells may be isolatedfrom biopsies, bone marrow, peripheral blood, or a fraction of bonemarrow or peripheral blood of a patient, using a commercially availablecell separation system, such as the Isolex™ System, available fromNexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No.5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO92/07243). In one particular embodiment of the present invention, Tcells may be isolated from intraepithelial lymphocytes (IEL) or laminapropria lymphocyte (LPL) samples originating from colon biopsies.Individuals with skill in the art will readily recognize that therenumerous methodologies for isolating IEL and LPL (for example, methodsdescribed in Christ, A. D., S. P. Colgan, S. P. Balk, R. S. Blumberg.1997. Immunol. Lett. 58:159; Boll G, Reimann J. Scand J Immunol 1995August; 42(2):191-201). In certain aspects, T cells may be derived fromrelated or unrelated humans, non-human mammals, cell lines or cultures.

[0299] T cells may be stimulated with a polypeptide, polynucleotideencoding a polypeptide and/or an antigen presenting cell (APC) thatexpresses such a polypeptide. Such stimulation is performed underconditions and for a time sufficient to permit the generation of T cellsthat are specific for the polypeptide of interest. Preferably, abacterial polypeptide or polynucleotide of the invention is presentwithin a delivery vehicle, such as a microsphere, to facilitate thegeneration of specific T cells.

[0300] T cells are considered to be specific for a polypeptide of thepresent invention if the T cells specifically proliferate, secretecytokines or kill target cells coated with the polypeptide or expressinga gene encoding the polypeptide. T cell specificity may be evaluatedusing any of a variety of standard techniques. For example, within achromium release assay or proliferation assay, a stimulation index ofmore than two fold increase in lysis and/or proliferation, compared tonegative controls, indicates T cell specificity. Such assays may beperformed, for example, as described in Chen et al., Cancer Res.54:1065-1070, 1994. Alternatively, detection of the proliferation of Tcells may be accomplished by a variety of known techniques. For example,T cell proliferation can be detected by measuring an increased rate ofDNA synthesis (e.g., by pulse-labeling cultures of T cells withtritiated thymidine and measuring the amount of tritiated thymidineincorporated into DNA). Contact with a bacterial polypeptide (100ng/ml-100 μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days willtypically result in at least a two fold increase in proliferation of theT cells. Contact as described above for 2-3 hours should result inactivation of the T cells, as measured using standard cytokine assays inwhich a two fold increase in the level of cytokine release (e.g., TNF orIFN-γ) is indicative of T cell activation (see Coligan et al., CurrentProtocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)). Tcells that have been activated in response to a bacterial polypeptide,polynucleotide or polypeptide-expressing APC may be CD4⁺ and/or CD8⁺.Bacterial polypeptide-specific T cells may be expanded using standardtechniques. Within preferred embodiments, the T cells are derived from apatient, a related donor or an unrelated donor, and are administered tothe patient following stimulation and expansion.

[0301] For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferatein response to a bacterial polypeptide, polynucleotide or APC can beexpanded in number either in vitro or in vivo. Proliferation of such Tcells in vitro may be accomplished in a variety of ways. For example,the T cells can be re-exposed to a bacterial polypeptide, or a shortpeptide corresponding to an immunogenic portion of such a polypeptide,with or without the addition of T cell growth factors, such asinterleukin-2, and/or stimulator cells that synthesize a bacterialpolypeptide. Alternatively, one or more T cells that proliferate in thepresence of the bacterial polypeptide can be expanded in number bycloning. Methods for cloning cells are well known in the art, andinclude limiting dilution.

[0302] In certain embodiments, T cells that produce anti-inflammatorycytokines may be desirable. Such cytokines may include, but are notlimited to, 10 (IL-10), interferon-γ (IFN-γ), interleukin 4 (IL-4),interleukin 12 (IL-12), transforming growth factor beta (TGFβ andinterleukin 18 (IL-18). In certain embodiments, an anti-inflammatoryresponse is mediated by CD4+ T helper cells.

[0303] T Cell Receptor Compositions

[0304] The T cell receptor (TCR) consists of 2 different, highlyvariable polypeptide chains, termed the T-cell receptor α and β chains,that are linked by a disulfide bond (Janeway, Travers, Walport.Immunobiology. Fourth Ed., 148-159. Elsevier Science Ltd/GarlandPublishing. 1999). The α/β heterodimer complexes with the invariant CD3chains at the cell membrane. This complex recognizes specific antigenicpeptides bound to MHC molecules. The enormous diversity of TCRspecificities is generated much like immunoglobulin diversity, throughsomatic gene rearrangement. The β chain genes contain over 50 variable(V), 2 diversity (D), over 10 joining (J) segments, and 2 constantregion segments (C). The a chain genes contain over 70 V segments, andover 60 J segments but no D segments, as well as one C segment. During Tcell development in the thymus, the D to J gene rearrangement of the βchain occurs, followed by the V gene segment rearrangement to the DJ.This functional VDJ_(β) exon is transcribed and spliced to join to aC_(β). For the a chain, a V_(α) gene segment rearranges to a J_(α) genesegment to create the functional exon that is then transcribed andspliced to the C_(α). Diversity is further increased during therecombination process by the random addition of P and N-nucleotidesbetween the V, D, and J segments of the β chain and between the V and Jsegments in the α chain (Janeway, Travers, Walport. Immunobiology.Fourth Ed., 98 and 150. Elsevier Science Ltd/Garland Publishing. 1999).

[0305] The present invention, in another aspect, provides TCRs specificfor a polypeptide disclosed herein, or for a variant or derivativethereof. In accordance with the present invention, polynucleotide andamino acid sequences are provided for the V-J or V-D-J junctionalregions or parts thereof for the alpha and beta chains of the T-cellreceptor which recognize bacterial polypeptides described herein. Ingeneral, this aspect of the invention relates to T-cell receptors whichrecognize or bind bacterial polypeptides presented in the context ofMHC. In a preferred embodiment the bacterial antigens recognized by theT-cell receptors comprise a polypeptide of the present invention. Forexample, cDNA encoding a TCR specific for a bacterial peptide can beisolated from T cells specific for a bacterial polypeptide usingstandard molecular biological and recombinant DNA techniques.

[0306] This invention further includes the T-cell receptors or analogsthereof having substantially the same function or activity as the T-cellreceptors of this invention which recognize or bind bacterialpolypeptides. Such receptors include, but are not limited to, a fragmentof the receptor, or a substitution, addition or deletion mutant of aT-cell receptor provided herein. This invention also encompassespolypeptides or peptides that are substantially homologous to the T-cellreceptors provided herein or that retain substantially the sameactivity. The term “analog” includes any protein or polypeptide havingan amino acid residue sequence substantially identical to the T-cellreceptors provided herein in which one or more residues, preferably nomore than 5 residues, more preferably no more than 25 residues have beenconservatively substituted with a functionally similar residue and whichdisplays the functional aspects of the T-cell receptor as describedherein.

[0307] The present invention further provides for suitable mammalianhost cells, for example, non-specific T cells, that are transfected witha polynucleotide encoding TCRs specific for a polypeptide describedherein, thereby rendering the host cell specific for the polypeptide.The α and β chains of the TCR may be contained on separate expressionvectors or alternatively, on a single expression vector that alsocontains an internal ribosome entry site (IRES) for cap-independenttranslation of the gene downstream of the IRES. Said host cellsexpressing TCRs specific for the polypeptide may be used, for example,for adoptive immunotherapy of IBD as discussed further below.

[0308] In further aspects of the present invention, cloned TCRs specificfor a polypeptide recited herein may be used in a kit for the diagnosisof IBD. For example, the nucleic acid sequence or portions thereof, ofbacterial-specific TCRs can be used as probes or primers for thedetection of expression of the rearranged genes encoding the specificTCR in a biological sample. Therefore, the present invention furtherprovides for an assay for detecting messenger RNA or DNA encoding theTCR specific for a polypeptide.

[0309] Pharmaceutical Compositions

[0310] In additional embodiments, the present invention concernsformulation of one or more of the polynucleotide, polypeptide, T-cell,TCR, and/or antibody compositions disclosed herein inpharmaceutically-acceptable carriers for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy.

[0311] It will be understood that, if desired, a composition asdisclosed herein may be administered in combination with other agents aswell, such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

[0312] Therefore, in another aspect of the present invention,pharmaceutical compositions are provided comprising one or more of thepolynucleotide, polypeptide, antibody, TCR, and/or T-cell compositionsdescribed herein in combination with a physiologically acceptablecarrier. In certain preferred embodiments, the pharmaceuticalcompositions of the invention comprise immunogenic polynucleotide and/orpolypeptide compositions of the invention for use in prophylactic andtheraputic vaccine applications. Vaccine preparation is generallydescribed in, for example, M. F. Powell and M. J. Newman, eds., “VaccineDesign (the subunit and adjuvant approach),” Plenum Press (NY, 1995).Generally, such compositions will comprise one or more polynucleotideand/or polypeptide compositions of the present invention in combinationwith one or more immunostimulants.

[0313] It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

[0314] In another embodiment, illustrative immunogenic compositions,e.g., vaccine compositions, of the present invention comprise DNAencoding one or more of the polypeptides as described above, such thatthe polypeptide is generated in situ. As noted above, the polynucleotidemay be administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

[0315] Therefore, in certain embodiments, polynucleotides encodingimmunogenic polypeptides described herein are introduced into suitablemammalian host cells for expression using any of a number of knownviral-based systems. In one illustrative embodiment, retrovirusesprovide a convenient and effective platform for gene delivery systems. Aselected nucleotide sequence encoding a polypeptide of the presentinvention can be inserted into a vector and packaged in retroviralparticles using techniques known in the art. The recombinant virus canthen be isolated and delivered to a subject. A number of illustrativeretroviral systems have been described (e.g., U.S. Pat. No. 5,219,740;Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990)Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852;Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; andBoris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[0316] In addition, a number of illustrative adenovirus-based systemshave also been described. Unlike retroviruses which integrate into thehost genome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

[0317] Various adeno-associated virus (AAV) vector systems have alsobeen developed for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

[0318] Additional viral vectors useful for delivering thepolynucleotides encoding polypeptides of the present invention by genetransfer include those derived from the pox family of viruses, such asvaccinia virus and avian poxvirus. By way of example, vaccinia virusrecombinants expressing the novel molecules can be constructed asfollows. The DNA encoding a polypeptide is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the polypeptideof interest into the viral genome. The resulting TK.sup.(−) recombinantcan be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0319] A vaccinia-based infection/transfection system can beconveniently used to provide for inducible, transient expression orcoexpression of one or more polypeptides described herein in host cellsof an organism. In this particular system, cells are first infected invitro with a vaccinia virus recombinant that encodes the bacteriophageT7 RNA polymerase. This polymerase displays exquisite specificity inthat it only transcribes templates bearing T7 promoters. Followinginfection, cells are transfected with the polynucleotide orpolynucleotides of interest, driven by a T7 promoter. The polymeraseexpressed in the cytoplasm from the vaccinia virus recombinanttranscribes the transfected DNA into RNA which is then translated intopolypeptide by the host translational machinery. The method provides forhigh level, transient, cytoplasmic production of large quantities of RNAand its translation products. See, e.g., Elroy-Stein and Moss, Proc.Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl.Acad. Sci. USA (1986) 83:8122-8126.

[0320] Alternatively, avipoxviruses, such as the fowlpox and canarypoxviruses, can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[0321] Any of a number of alphavirus vectors can also be used fordelivery of polynucleotide compositions of the present invention, suchas those vectors described in U.S. Pat. Nos. 5,843,723; 6,015,686;6,008,035 and 6,015,694. Certain vectors based on Venezuelan EquineEncephalitis (VEE) can also be used, illustrative examples of which canbe found in U.S. Pat. Nos. 5,505,947 and 5,643,576.

[0322] Moreover, molecular conjugate vectors, such as the adenoviruschimeric vectors described in Michael et al. J. Biol. Chem. (1993)268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992)89:6099-6103, can also be used for gene delivery under the invention.

[0323] Additional illustrative information on these and other knownviral-based delivery systems can be found, for example, in Fisher-Hochet al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al.,Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21,1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973;U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805;Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219,1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502,1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al.,Cir. Res. 73:1202-1207, 1993.

[0324] In certain embodiments, a polynucleotide may be integrated intothe genome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

[0325] In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

[0326] In still another embodiment, a composition of the presentinvention can be delivered via a particle bombardment approach, many ofwhich have been described. In one illustrative example, gas-drivenparticle acceleration can be achieved with devices such as thosemanufactured by Powderject Pharmaceuticals PLC (Oxford, UK) andPowderject Vaccines Inc. (Madison, Wis.), some examples of which aredescribed in U.S. Pat. Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807;and EP Patent No. 0500 799. This approach offers a needle-free deliveryapproach wherein a dry powder formulation of microscopic particles, suchas polynucleotide or polypeptide particles, are accelerated to highspeed within a helium gas jet generated by a hand held device,propelling the particles into a target tissue of interest.

[0327] In a related embodiment, other devices and methods that may beuseful for gas-driven needle-less injection of compositions of thepresent invention include those provided by Bioject, Inc. (Portland,Oreg.), some examples of which are described in U.S. Pat. Nos.4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and5,993,412.

[0328] According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, antibody, T-cell, TCR,and/or APC compositions of this invention. An immunostimulant refers toessentially any substance that enhances or potentiates an immuneresponse (antibody and/or cell-mediated) to an exogenous antigen. Onepreferred type of immunostimulant comprises an adjuvant. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

[0329] Within certain embodiments of the invention, the adjuvantcomposition is preferably one that induces an anti-inflammatory immuneresponse (antibody or cell-mediated). Accordingly, high levels ofanti-inflammatory cytokines (anti-inflammatory cytokines may include,but are not limited to, interleukin 4 (IL-4), interleukin 5 (IL-5),interleukin 10 (IL-10), and transforming growth factor beta (TGFβ wouldbe preferred. In certain embodiments, an anti-inflammatory responsewould be mediated by CD4+ T helper cells. Bacterial flagellin have beensuggested to act as adjunvants (McSorley et al., J. Immunol.169:3914-19, 2002). Within one embodiment of the invention, theflagellin proteins disclosed herein can be used in adjuvantcompositions.

[0330] Within other embodiments, the adjuvants used in conjunction withthe compositions of the present invention increase lipopolysaccharide(LPS) responsiveness. Illustrative adjuvants include but are not limitedto, monophosphoryl lipid A (MPL), aminoalkyl glucosaminide 4-phosphates(AGPs), including RC-512, RC-522, RC-527, RC-529, RC-544, and RC-560(Corixa, Hamilton, Mont.) and other AGPs such as those described inpending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties.

[0331] Within other embodiments of the invention, the adjuvantcomposition is one that induces an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989. Alternatively, in a related embodiment, inwhich a preferred response is predominantly Th2-type, the level ofTh2-type cytokines will increase to a greater extent than the level ofTh1-type cytokines. Again, the levels of these cytokines may be readilyassessed using standard assays.

[0332] Certain preferred adjuvants for eliciting a predominantlyTh1-type response include, for example, a combination of monophosphoryllipid A, preferably 3-de-O-acylated monophosphoryl lipid A, togetherwith an aluminum salt. MPL® adjuvants are available from CorixaCorporation (Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727;4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (inwhich the CpG dinucleotide is unmethylated) also induce a predominantlyTh1 response. Such oligonucleotides are well known and are described,for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200and 5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

[0333] Alternatively the saponin formulations may be combined withvaccine vehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

[0334] In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

[0335] Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

[0336] Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montamide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

[0337] Other preferred adjuvants include adjuvant molecules of thegeneral formula

HO(CH₂CH₂O)_(n)-A-R,  (I)

[0338] wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl orPhenyl C₁₋₅₀ alkyl.

[0339] One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

[0340] The polyoxyethylene ether according to the general formula (I)above may, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

[0341] According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-bacterial effects perse and/or to be immunologically compatible with the receiver (i.e.,matched HLA haplotype). APCs may generally be isolated from any of avariety of biological fluids and organs, including bacterial andperibacterial tissues, and may be autologous, allogeneic, syngeneic orxenogeneic cells.

[0342] Certain preferred embodiments of the present invention usedendritic cells or progenitors thereof as antigen-presenting cells.Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantibacterial immunity (see Timmerman and Levy, Ann. Rev. Med.50:507-529, 1999). In general, dendritic cells may be identified basedon their typical shape (stellate in situ, with marked cytoplasmicprocesses (dendrites) visible in vitro), their ability to take up,process and present antigens with high efficiency and their ability toactivate naïve T cell responses. Dendritic cells may, of course, beengineered to express specific cell-surface receptors or ligands thatare not commonly found on dendritic cells in vivo or ex vivo, and suchmodified dendritic cells are contemplated by the present invention. Asan alternative to dendritic cells, secreted vesicles antigen-loadeddendritic cells (called exosomes) may be used within a vaccine (seeZitvogel et al., Nature Med. 4:594-600, 1998).

[0343] Dendritic cells and progenitors may be obtained from peripheralblood, bone marrow, tumor-infiltrating cells, peritumoraltissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cordblood or any other suitable tissue or fluid. For example, dendriticcells may be differentiated ex vivo by adding a combination of cytokinessuch as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytesharvested from peripheral blood. Alternatively, CD34 positive cellsharvested from peripheral blood, umbilical cord blood or bone marrow maybe differentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

[0344] Dendritic cells are conveniently categorized as “immature” and“mature” cells, which allows a simple way to discriminate between twowell characterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

[0345] APCs may generally be transfected with a polynucleotide of theinvention (or portion or other variant thereof) such that the encodedpolypeptide, or an immunogenic portion thereof, is expressed on the cellsurface. Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the bacterial polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

[0346] While any suitable carrier known to those of ordinary skill inthe art may be employed in the pharmaceutical compositions of thisinvention, the type of carrier will typically vary depending on the modeof administration. Compositions of the present invention may beformulated for any appropriate manner of administration, including forexample, topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

[0347] Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

[0348] In another illustrative embodiment, biodegradable microspheres(e.g., polylactate polyglycolate) are employed as carriers for thecompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and5,942,252. Modified hepatitis B core protein carrier systems, such asdescribed in WO/99 40934, and references cited therein, will also beuseful for many applications. Another illustrative carrier/deliverysystem employs a carrier comprising particulate-protein complexes, suchas those described in U.S. Pat. No. 5,928,647, which are capable ofinducing a class I-restricted cytotoxic T lymphocyte responses in ahost.

[0349] In another illustrative embodiment, calcium phosphate coreparticles are employed as carriers, vaccine adjuvants, or as controlledrelease matrices for the compositions of this invention. Exemplarycalcium phosphate particles are disclosed, for example, in publishedpatent application No. WO/0046147.

[0350] The pharmaceutical compositions of the invention will oftenfurther comprise one or more buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, bacteriostats, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes thatrender the formulation isotonic, hypotonic or weakly hypertonic with theblood of a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

[0351] The pharmaceutical compositions described herein may be presentedin unit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

[0352] The development of suitable dosing and treatment regimens forusing the particular compositions described herein in a variety oftreatment regimens, including e.g., oral, parenteral, intravenous,intranasal, and intramuscular administration and formulation, is wellknown in the art, some of which are briefly discussed below for generalpurposes of illustration.

[0353] In certain applications, the pharmaceutical compositionsdisclosed herein may be delivered via oral administration to an animal.As such, these compositions may be formulated with an inert diluent orwith an assimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

[0354] The active compounds may even be incorporated with excipients andused in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like (see, forexample, Mathiowitz et al., Nature Mar. 27, 1997; 386(6623):410-4; Hwanget al., Crit Rev Ther Drug Carrier Syst 1998; 15(3):243-84; U.S. Pat.No. 5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

[0355] Typically, these formulations will contain at least about 0.1% ofthe active compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

[0356] For oral administration the compositions of the present inventionmay alternatively be incorporated with one or more excipients in theform of a mouthwash, dentifrice, buccal tablet, oral spray, orsublingual orally-administered formulation. Alternatively, the activeingredient may be incorporated into an oral solution such as onecontaining sodium borate, glycerin and potassium bicarbonate, ordispersed in a dentifrice, or added in a therapeutically-effectiveamount to a composition that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants. Alternatively thecompositions may be fashioned into a tablet or solution form that may beplaced under the tongue or otherwise dissolved in the mouth.

[0357] In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

[0358] Illustrative pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

[0359] In one embodiment, for parenteral administration in an aqueoussolution, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, a sterile aqueous medium that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. Moreover, for humanadministration, preparations will of course preferably meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

[0360] In another embodiment of the invention, the compositionsdisclosed herein may be formulated in a neutral or salt form.Illustrative pharmaceutically-acceptable salts include the acid additionsalts (formed with the free amino groups of the protein) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective.

[0361] The carriers can further comprise any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. The phrase“pharmaceutically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human.

[0362] In certain embodiments, the pharmaceutical compositions may bedelivered by intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs via nasal aerosol sprays hasbeen described, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No.5,804,212. Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., J Controlled Release Mar. 2,1998; 52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.S. Pat.No. 5,725,871) are also well-known in the pharmaceutical arts. Likewise,illustrative transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045.

[0363] In certain embodiments, liposomes, nanocapsules, microparticles,lipid particles, vesicles, and the like, are used for the introductionof the compositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

[0364] The formation and use of liposome and liposome-like preparationsas potential drug carriers is generally known to those of skill in theart (see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995; 12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

[0365] Liposomes have been used successfully with a number of cell typesthat are normally difficult to transfect by other procedures, includingT cell suspensions, primary hepatocyte cultures and PC 12 cells(Renneisen et al., J Biol. Chem. Sep. 25, 1990; 265(27):16337-42; Mulleret al., DNA Cell Biol. 1990 April; 9(3):221-9). In addition, liposomesare free of the DNA length constraints that are typical of viral-baseddelivery systems. Liposomes have been used effectively to introducegenes, various drugs, radiotherapeutic agents, enzymes, viruses,transcription factors, allosteric effectors and the like, into a varietyof cultured cell lines and animals. Furthermore, he use of liposomesdoes not appear to be associated with autoimmune responses orunacceptable toxicity after systemic delivery.

[0366] In certain embodiments, liposomes are formed from phospholipidsthat are dispersed in an aqueous medium and spontaneously formmultilamellar concentric bilayer vesicles (also termed multilamellarvesicles (MLVs).

[0367] Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev India Pharm. 1998 December; 24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March;45(2):149-55; Zambaux et al. J Controlled Release. Jan. 2, 1998;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

[0368] Therapeutic Methods for IBD

[0369] Immunologic approaches to IBD therapy are based on therecognition that IIBD represents an “abnormal” mucosal immune responseto bacteria within the lumen of the gastrointestinal tract. The precisemolecular nature of the bacterial antigen(s) recognized by the immunesystem has not been described.

[0370] IBD immunotherapy generally focuses on inducing humoral immuneresponses, cellular immune responses, or both, with the goal of inducingtolerance to a particular enteric bacterial antigen, thereby leading toa decrease in inflammation in the gut. Moreover, induction of CD4⁺ Thelper cells is necessary in order to secondarily induce eitherantibodies or cytotoxic CD8⁺ T cells. Polypeptide antigens that areselective or ideally specific to IBD-associated bacteria offer apowerful approach for inducing anti-inflammatory immune responses thateither prevent or ameliorate an aberrant immune response to bacterialantigens associated with IBD, and are an important aspect of the presentinvention.

[0371] Therefore, in further aspects of the present invention, thepharmaceutical compositions described herein may be used to stimulate animmune response against bacterial antigens associated with IBD. In oneembodiment of the present invention, the immune response inducedcomprises antibodies that block the interaction of a bacterial antigenwith a host receptor. In one particular embodiment, antibodies inducedby the compositions of the present invention block the interactionbetween flagellin and TLR5, thereby ameliorating the pro-inflammatorycascade initiated by NFKB activation. Alternatively, the compositions ofthe present invention would induce antibodies that stimulateresponsiveness to LPS that ameliorated the hypo-responsiveness inindividuals with Nod2 gene mutation associated with IBD.

[0372] In a further embodiment of the present invention, an immuneresponse would be anti-inflammatory in nature. For example, a cellularimmune response wherein the T cells produce anti-inflammatory cytokines.Anti-inflammatory cytokines may include, but are not limited to, IL-4,IL-5, IL-10, TGF-β.

[0373] Within such methods, the pharmaceutical compositions describedherein are administered to a patient, typically a warm-blooded animal,preferably a human. A patient may or may not be afflicted with IBD. Asdiscussed above, administration of the pharmaceutical compositions maybe by any suitable method, including administration by intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal,anal, vaginal, topical and oral routes.

[0374] Within certain embodiments, immunotherapy may be activeimmunotherapy, in which treatment relies on the in vivo stimulation ofthe endogenous host immune system to react against bacteria with theadministration of immune response-modifying agents or immunomodulators(such as polypeptides and polynucleotides as provided herein).

[0375] Within other embodiments, immunotherapy may be passiveimmunotherapy, in which treatment involves the delivery of agents withestablished antibacterial immune reactivity (such as effector cells orantibodies) that can directly or indirectly mediate antibacterialeffects and does not necessarily depend on an intact host immune system.Examples of effector cells include T cells as discussed above, Tlymphocytes (such as CD8⁺ cytotoxic T lymphocytes and CD4⁺ T-helperlymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

[0376] Monoclonal antibodies may be labeled with any of a variety oflabels for desired selective usages in detection, diagnostic assays ortherapeutic applications (as described in U.S. Pat. Nos. 6,090,365;6,015,542; 5,843,398; 5,595,721; and 4,708,930, hereby incorporated byreference in their entirety as if each was incorporated individually).In each case, the binding of the labelled monoclonal antibody to thedeterminant site of the antigen will signal detection or delivery of aparticular therapeutic agent to the antigenic determinant on thenon-normal cell. A further object of this invention is to provide thespecific monoclonal antibody suitably labelled for achieving suchdesired selective usages thereof.

[0377] Effector cells may generally be obtained in sufficient quantitiesfor adoptive immunotherapy by growth in vitro, as described herein.Culture conditions for expanding single antigen-specific effector cellsto several billion in number with retention of antigen recognition invivo are well known in the art. Such in vitro culture conditionstypically use intermittent stimulation with antigen, often in thepresence of cytokines (such as IL-2) and non-dividing feeder cells. Asnoted above, immunoreactive polypeptides as provided herein may be usedto rapidly expand antigen-specific T cell cultures in order to generatea sufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

[0378] Alternatively, a vector expressing a polypeptide recited hereinmay be introduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal administration.

[0379] Routes and frequency of administration of the therapeuticcompositions described herein, as well as dosage, will vary fromindividual to individual, and may be readily established using standardtechniques. In general, the pharmaceutical compositions and vaccines maybe administered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an appropriate anti-bacterialimmune response, and is at least 10-50% above the basal (i.e.,untreated) level. Alternatively, a suitable dose is an amount of acompound that, when administered as described above, is capable ofdecreasing inflammation in the colon associated with IBD. Such adecrease is at least 10-50% below the untreated level. Such response canbe monitored by measuring the anti-bacterial antibodies in a patient orby vaccine-dependent generation of cytolytic effector cells capable ofrecognizing in vitro bacterial antigens identified from bacteriaisolated from the patient. Such vaccines should also be capable ofcausing an immune response that leads to an improved clinical outcome(e.g., more frequent remissions, complete or partial or longerdisease-free survival) in vaccinated patients as compared tonon-vaccinated patients. In general, for pharmaceutical compositions andvaccines comprising one or more polypeptides, the amount of eachpolypeptide present in a dose ranges from about 25 μg to 5 mg per kg ofhost. Suitable dose sizes will vary with the size of the patient, butwill typically range from about 0.1 mL to about 5 mL.

[0380] In general, an appropriate dosage and treatment regimen providesthe active compound(s) in an amount sufficient to provide therapeuticand/or prophylactic benefit. Such a response can be monitored byestablishing an improved clinical outcome (e.g., a decrease ininflammation in the gut, decrease in diarrhea, decrease in steroidrequirements or requirement for other immunosuppressive therapies,decrease in anemia, or decrease in Crohn's disease activity index(CDAI)) in treated patients as compared to non-treated patients.Increases in appropriate anti-inflammatory immune responses to abacterial protein generally correlate with an improved clinical outcome.Such immune responses may generally be evaluated using standardproliferation, cytotoxicity or cytokine assays, which may be performedusing samples obtained from a patient before and after treatment.

[0381] Detection and Diagnostic Compositions, Methods and Kits

[0382] There are a variety of assay formats known to those of ordinaryskill in the art for using a binding agent to detect polypeptide markersin a sample. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In general, the presence orabsence of an IBD-associated bacteria in a patient may be determined by(a) contacting a biological sample obtained from a patient with abinding agent; (b) detecting in the sample a level of polypeptide thatbinds to the binding agent; and (c) comparing the level of polypeptidewith a predetermined cut-off value.

[0383] In a preferred embodiment, the assay involves the use of bindingagent immobilized on a solid support to bind to and remove thepolypeptide from the remainder of the sample. The bound polypeptide maythen be detected using a detection reagent that contains a reportergroup and specifically binds to the binding agent/polypeptide complex.Such detection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length bacterial proteins and polypeptide portions thereof to whichthe binding agent binds, as described above.

[0384] The solid support may be any material known to those of ordinaryskill in the art to which the bacterial protein may be attached. Forexample, the solid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

[0385] Covalent attachment of binding agent to a solid support maygenerally be achieved by first reacting the support with a bifunctionalreagent that will react with both the support and a functional group,such as a hydroxyl or amino group, on the binding agent. For example,the binding agent may be covalently attached to supports having anappropriate polymer coating using benzoquinone or by condensation of analdehyde group on the support with an amine and an active hydrogen onthe binding partner (see, e.g., Pierce Immunotechnology Catalog andHandbook, 1991, at A12-A13).

[0386] In certain embodiments, the assay is a two-antibody sandwichassay. This assay may be performed by first contacting an antibody thathas been immobilized on a solid support, commonly the well of amicrotiter plate, with the sample, such that polypeptides within thesample are allowed to bind to the immobilized antibody. Unbound sampleis then removed from the immobilized polypeptide-antibody complexes anda detection reagent (preferably a second antibody capable of binding toa different site on the polypeptide) containing a reporter group isadded. The amount of detection reagent that remains bound to the solidsupport is then determined using a method appropriate for the specificreporter group.

[0387] More specifically, once the antibody is immobilized on thesupport as described above, the remaining protein binding sites on thesupport are typically blocked. Any suitable blocking agent known tothose of ordinary skill in the art, such as bovine serum albumin orTween 20™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibodyis then incubated with the sample, and polypeptide is allowed to bind tothe antibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of an IBD-associated bacterialpolypeptide within a sample obtained from an individual with IBD atleast about 95% of that achieved at equilibrium between bound andunbound polypeptide. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 minutes is generallysufficient.

[0388] Unbound sample may then be removed by washing the solid supportwith an appropriate buffer, such as PBS containing 0.1% Tween 20™. Thesecond antibody, which contains a reporter group, may then be added tothe solid support. Preferred reporter groups include those groupsrecited above.

[0389] The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

[0390] To determine the presence or absence of IBD, the signal detectedfrom the reporter group that remains bound to the solid support isgenerally compared to a signal that corresponds to a predeterminedcut-off value. In one preferred embodiment, the cut-off value for thedetection of an IBD-associated bacterial antigen is the average meansignal obtained when the immobilized antibody is incubated with samplesfrom patients without IBD. In general, a sample generating a signal thatis three standard deviations above the predetermined cut-off value isconsidered positive for IBD-associated bacterial antigens. In analternate preferred embodiment, the cut-off value is determined using aReceiver Operator Curve, according to the method of Sackett et al.,Clinical Epidemiology: A Basic Science for Clinical Medicine, LittleBrown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-offvalue may be determined from a plot of pairs of true positive rates(i.e., sensitivity) and false positive rates (100%-specificity) thatcorrespond to each possible cut-off value for the diagnostic testresult. The cut-off value on the plot that is the closest to the upperleft-hand corner (i.e., the value that encloses the largest area) is themost accurate cut-off value, and a sample generating a signal that ishigher than the cut-off value determined by this method may beconsidered positive. Alternatively, the cut-off value may be shifted tothe left along the plot, to minimize the false positive rate, or to theright, to minimize the false negative rate. In general, a samplegenerating a signal that is higher than the cut-off value determined bythis method is considered positive for an IBD-associated bacteria.

[0391] In a related embodiment, the assay is performed in a flow-throughor strip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of an IBD-associated bacterial antigen. Typically, theconcentration of second binding agent at that site generates a pattern,such as a line, that can be read visually. The absence of such a patternindicates a negative result. In general, the amount of binding agentimmobilized on the membrane is selected to generate a visuallydiscernible pattern when the biological sample contains a level ofpolypeptide that would be sufficient to generate a positive signal inthe two-antibody sandwich assay, in the format discussed above.Preferred binding agents for use in such assays are antibodies andantigen-binding fragments thereof. Preferably, the amount of antibodyimmobilized on the membrane ranges from about 25 ng to about 1 μg, andmore preferably from about 50 ng to about 500 ng. Such tests cantypically be performed with a very small amount of biological sample.

[0392] Of course, numerous other assay protocols exist that are suitablefor use with the bacterial proteins or binding agents of the presentinvention. The above descriptions are intended to be exemplary only. Forexample, it will be apparent to those of ordinary skill in the art thatthe above protocols may be readily modified to use bacterialpolypeptides to detect antibodies that bind to such polypeptides in abiological sample. The detection of such bacterial protein specificantibodies may correlate with the presence of an IBD-associated antigen.

[0393] IBD-associated bacteria may also, or alternatively, be detectedbased on the presence of T cells that specifically react with abacterial protein in a biological sample. Within certain methods, abiological sample comprising CD4⁺ and/or CD8⁺ T cells isolated from apatient is incubated with a bacterial polypeptide, a polynucleotideencoding such a polypeptide and/or an APC that expresses at least animmunogenic portion of such a polypeptide, and the presence or absenceof specific activation of the T cells is detected. Suitable biologicalsamples include, but are not limited to, isolated T cells. For example,T cells may be isolated from a patient by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes). In one particular embodiment of the present invention, Tcells may be isolated from intraepithelial lymphocytes (IEL) or laminapropria lymphocyte (LPL) samples originating from colon biopsies. Tcells may be incubated in vitro for 2-9 days (typically 4 days) at 37□Cwith polypeptide (e.g., 5-25 μg/ml). It may be desirable to incubateanother aliquot of a T cell sample in the absence of bacterialpolypeptide to serve as a control. For CD4⁺ T cells, activation ispreferably detected by evaluating proliferation of the T cells. For CD8⁺T cells, activation is preferably detected by evaluating cytolyticactivity. A level of proliferation that is at least two fold greaterand/or a level of cytolytic activity that is at least 20% greater thanin disease-free patients indicates the presence of IBD-associatedbacteria in the patient.

[0394] As noted above, IBD may also, or alternatively, be detected basedon the level of mRNA encoding a bacterial protein in a biologicalsample. For example, at least two oligonucleotide primers may beemployed in a polymerase chain reaction (PCR) based assay to amplify aportion of a bacterial cDNA derived from a biological sample, wherein atleast one of the oligonucleotide primers is specific for (i.e.,hybridizes to) a polynucleotide encoding the bacterial protein. Theamplified cDNA is then separated and detected using techniques wellknown in the art, such as gel electrophoresis.

[0395] Similarly, oligonucleotide probes that specifically hybridize toa polynucleotide encoding a bacterial protein may be used in ahybridization assay to detect the presence of polynucleotide encodingthe bacterial protein in a biological sample.

[0396] To permit hybridization under assay conditions, oligonucleotideprimers and probes should comprise an oligonucleotide sequence that hasat least about 60%, preferably at least about 75% and more preferably atleast about 90%, identity to a portion of a polynucleotide encoding abacterial protein of the invention that is at least 10 nucleotides, andpreferably at least 20 nucleotides, in length. Preferably,oligonucleotide primers and/or probes hybridize to a polynucleotideencoding a polypeptide described herein under moderately stringentconditions, as defined above. Oligonucleotide primers and/or probeswhich may be usefully employed in the diagnostic methods describedherein preferably are at least 10-40 nucleotides in length. In apreferred embodiment, the oligonucleotide primers comprise at least 10contiguous nucleotides, more preferably at least 15 contiguousnucleotides, of a DNA molecule having a sequence as disclosed herein.Techniques for both PCR based assays and hybridization assays are wellknown in the art (see, for example, Mullis et al., Cold Spring HarborSymp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, StocktonPress, NY, 1989).

[0397] One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

[0398] In another embodiment, the compositions described herein may beused as markers for the progression of IBD. In this embodiment, assaysas described above for the diagnosis of IBD may be performed over time,and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, IBD is progressing in those patients inwhom the level of polypeptide or polynucleotide detected increases overtime. In contrast, IBDis not progressing when the level of reactivepolypeptide or polynucleotide either remains constant or decreases withtime.

[0399] In a further embodiment, the compositions described herein may beused to monitor the level of antibodies specific for and/or T cellresponsiveness to an IBD-associated bacterial protein as a measure ofIBD progression. In general, IBD is progressing in those patients inwhom the level of antibodies that bind to a polypeptide or encoded by apolynucleotide described herein, that are detected increases over time.In contrast, IBD is not progressing when the level of reactiveantibodies either remains constant or decreases with time.

[0400] Certain in vivo diagnostic assays may be performed directly on alesion in the colon. One such assay involves contacting cells from alesion with a binding agent. The bound binding agent may then bedetected directly or indirectly via a reporter group. Such bindingagents may also be used in histological applications. Alternatively,polynucleotide probes may be used within such applications.

[0401] As noted above, to improve sensitivity, multiple bacterialproteins may be assayed within a given sample. It will be apparent thatbinding agents specific for different proteins, antibodies, or T cellsspecific thereto provided herein may be combined within a single assay.Further, multiple primers or probes may be used concurrently. Theselection of bacterial proteins may be based on routine experiments todetermine combinations that results in optimal sensitivity. In addition,or alternatively, assays for bacterial proteins, antibodies, or T cellsspecific thereto, provided herein may be combined with assays for otherknown bacterial antigens or genetic markers such as the NOD2 mutation.

[0402] The present invention further provides kits for use within any ofthe above diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a bacterial protein. Such antibodiesor fragments may be provided attached to a support material, asdescribed above. One or more additional containers may enclose elements,such as reagents or buffers, to be used in the assay. Such kits mayalso, or alternatively, contain a detection reagent as described abovethat contains a reporter group suitable for direct or indirect detectionof antibody binding.

[0403] Alternatively, a kit may be designed to detect the level of mRNAencoding a bacterial protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a bacterial protein.Such an oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding abacterial protein.

[0404] In an alternative embodiment, a kit may be designed to detect thelevel of antibodies specific for an IBD-associated bacterial protein ina biological sample.

[0405] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 IDENTIFICATION OF IBD-ASSOCIATED BACTERIAL ANTIGENSFROM A MOUSE CECAL BACTERIA GENOMIC RANDOM SHEAR EXPRESSION LIBRARY

[0406] A mouse cecal bacteria genomic random shear expression librarywas constructed by sonicating C3H/HeJ Bir mouse cecal bacteria genomicDNA to produce fragment sizes of approximately 0.1 to 5.0 kbp. 14 μg ofsonicated DNA was treated with DNA polymerase I, Klenow fragment, for 30minutes followed by Pfu polymerase for 30 minutes to produce blunt endedfragments. EcoRI adaptors were then ligated to the fragments and thenadaptors were phosphorylated with E. coli polynucleotide kinase.Fragments were next fractionated with a Sephacryl S400 column andfinally ligated to a Lambda ZAP Express (Stratagene) vector. Ligatedvector was then packaged with Gigapack III Gold packaging extract(Stratagene) and the unamplified library was plated with host E. coliXL-1 Blue MRF′ cells on LB agarose plates at a concentration of 25,000plaque forming units (PFU) per plate for 15 plates. After incubation at42° C. for 4 hours, nitrocellulose filters, soaked in 10 mM IPTG, wereadded and the plates which were incubated at 37° C. over night. Filterswere removed and washed 3× with PBS containing 0.1% Tween 20 (PBST),blocked for 1 hour with 1% BSA in PBST, washed 3× with PBST and thenincubated overnight at 4° C. in serum, preadsorbed with E. coliproteins, from C3H/HeJ Bir mice with inflammatory bowel disease. Afterwashing 3× with PBST, filters were incubated in a goat anti-mouse IgG,IgA, IgM secondary antibody conjugated with alkaline phosphatase for 1hour at room temperature. Filters were finally washed 3× with PBST, 2×with alkaline phosphatase buffer and developed with BCIP/NBT. Positiveclones were purified using the same technique; phagemid was excised; andresulting plasmid DNA was sequenced and searched against the GenBankdatabases. Those sequences that showed some degree of similarity toknown sequences in the database are listed in Table 2. Those sequencesthat showed no significant similarity to known sequences in the databaseare shown in Table 3. TABLE 2 BACTERIAL SEQUENCES THAT SHOWED SOMEDEGREE OF SIMILARITY TO KNOWN SEQUENCES IN THE DATABASE cDNA PRO SEQ SEQClone Clone Insert ID ID Name ID kbp Blastn Blastx 1 38 Cbir-1 76779 1.3No match (73%) flagellin A protein / (48%) FlaB [Butyrivibriofibrisolvens] (AF026812) 2 39 Cbir-2 76780 0.4 No match (73%)3-isopropylmalate dehydrogenase VC2491- Vibrio cholerae (group O1 strainN16961) 3 40, Cbir-3 76959 0.8 No match (53%) motility protein A- 41Termotoga maritima (strainMSB8) 5 Cbir-5 76961 1.2 No match (44%)methyl-accepting chemotaxis protein [Bacillushalodurans] (AP001520) 6 42Cbir-6 76781 1.0 No match (56%) ribosomal protein L6 (BL10) 7 Cbir-876962 3.0 No match (60%) acetohydroxy acid synthase large chain-Brevibacteriumflavum 10 Cbir-12 76964 2.0 (82%) (58%)phosphoenolpyruvate Same as carboxykinase VC2738- Blastx Vibriocholerae(group O1 strain N16961) 12 Cbir-14 76965 1.7 No match (49%) gapregulator [Staphylococcus aureus] (AJ133520) 13 Cbir-15 76966 1.0 (82%)(72%) flagellin A protein / Same as FlaB (Butyrivibrio Blastxfibrisolvens) (AF026812) 14 44 Cbir-16 76967 1.2 No match (58%)ribosomal protein L6 (BL10) 15 Cbir-18 76968 0.8 No match (69%)flagellin - Roseburia cecicola (65%) flagellin A protein / (63%) FlaB[Butyrivibrio fibrisolvens] (AF026812) 16 Cbir-19 77530 1.6 No match(51%) ABC-type transport protein-Synechocystis sp. (strain PCC6803) 1745 Cbir-20 76969 2.0 No match (68%) FlaB [Butyrivibrio fibrisolvens](AF026812) 18 Cbir-23 76970 0.8 (80%) (84%) elongation factor-Tu Same as[Porphyromonasgingivalis] Blastx (AB035462) 23 47 Cbir-32 76974 65bp Nomatch probably amidohydrolase Campylobacter jenjuni (strain NCTC 11168)24 Cbir-36 77074 1.6 (87%) (83%) elongation factor TU- Bacillus 1(Streptomyces subtilis ramocissimus) complete genome 26 48 Cbir-39 769750.6 No match (70%) Thermotoga maritima (strainMSB8) (55%) flagellin Aprotein / (56%) FlaB [Butyrivibrio fibrisolvens] (AF026812) 27 Cbir-4077075 0.7 No match (54%) flagellin-Thermotoga maritima (strain MSB8)/(58%) flagellin (Bacillus halodurans) (AP001512) 29 49 Cbir-44 76977 1.0No match (45%) flagellin-Thermotoga maritima (strainMSB8) / (57%)flagellin (Bacillus halodurans) (AP001512) 31 Cbir-46 77533 2.2 No match(AL512975) 32 Cbir-49 77534 0.9 No match (59%) flagellin-Roseburiacecicola (56%) flagellin A protein / (58%) FlaB (Butyrivibriofibriosolvens) (AF026812) 35 Cbir-62 77536 0.6 No match hemolysinsecretion protein precursor-Helecobacter pylori (strain 26695)/methyl-accepting chemotaxis protein (Helecobacter pylori) 36 Cbir-7377538 2.0 No match elongation factor - TS 37 Cbir-78 77539 1.0 No match(62%) flagellin-Roseburia cecicola (M20983) (57%) flagellin A protein /(59%) FlaB (Butyrivibrio fibrisolvens) 51 CB1-T2 73261 150 No match 36%Streptomyces Arabinosidase Secreted 54 CB1-T5 73264 1050 No match 33%Arabidopsis ClpC protease 55 CB1-T7 73266 650 No match 54% S. cerevisiaeAcetyltransferase 56 CB1-T8 73267 350 No match 28% Bacillus subtilisUnknown 58 CB1-T10 73269 150 No match ? 40% Deinococcus ABC transporter59 CB1-T11 73270 1700 No match 52% Borrelia burgdorferi Fructose-6-p 1-phosphotransferase 60 CB1-T13 73272 250 No match 56% Vibrio/Clostridiumalcohol dehydrogenase 61 CB1-T14 73273 260 No match ? 53% Bacteroidessurface Ag BspA 62 CB1-T15 73274 1000 No match 61% Clostridium prolyltRNA synthetase 64 CB3-T1 75037 1.7 ? 88% 65% Bacillus subtilis streptomoligopeptide transport ATP- yces bind pro OPPF 67 CB3-T4 75040 ? >600 Nomatch 36% E.coli/Staph Hypothetical 14.8 kDa membrane protein 70 CB3-T975044 300 No match 91% Helicobacter pylolri 2,3,4,5-tetrahydropyridine-2-carboxylaten- succinyltransferase 72 CB3-T12 75046 1700 No match 52%Thermotoga/Archaeoglobus/ Halobacterium conserved prot. 74 CB3-T14 750481300 No match 29% Streptomyces heat shock

[0407] TABLE 3 BACTERIAL SEQUENCES ThAT SHOWED NO SIGNIFICANT SIMILARITYTO KNOWN SEQUENCES IN THE DATABASE CDNA PRO SEQ SEQ Clone Clone InsertID ID Name ID kbp Blastn Blastx 4 Cbir-4 76960 2.1 No match No match 843 Cbir-9 76782 0.6 No match No match 9 Cbir-11 76963 1.5 No match Nomatch 11 Cbir-13 77529 1.5 No match No match 19 Cbir-24 76971 2.3 Nomatch No match 20 Cbir-26 77073 1.2 No match No match 21 46 Cbir-2776972 0.5 No match No match 22 Cbir-30 76973 0.7 No match No match 25Cbir-37 77531 2.5 No match No match 28 Cbir-41 76976 1.2 No match Nomatch 30 Cbir-45 77532 1.2 No match No match 33 Cbir-50 77535 0.7 Nomatch No match 34 50 Cbir-61 77076 0.5 No match No match 52 CB1-T3 73262600 No match No match 53 CB1-T4 73263 250 No match No match 57 CB1-T973268 250 No match No match 63 CB1-T16 73275 400 No match No match 65CB3-T2 75038 500 No match No match 66 CB3-T3 75039 500 No match No match68 CB3-T5 75041 400 No match No match 69 CB3-T6 75042 600 No match Nomatch 71 CB3-T10 75045 ? >500 No match No match 73 CB3-T13 75047 700 Nomatch No match

Example 2 CLONING AND EXPRESSION OF FLAGELLIN X

[0408] A novel flagellin was cloned by PCR amplification from totalcecal bacterium genomic DNA obtained from C3H/HeJ Bir mice.Oligonucleotide primers were developed with sequence from the aminoterminus of clone Cbir-1 (SEQ ID NO:1) and with a consensus sequencederived from the carboxy terminus of three flagellin sequences that aremost closely related the Cbir-1 sequence. A six histidine tag and a NdeI endonuclease cleavage site were added to the amino primer for cloningand for purification of recombinant protein and a Hind III cloning sitewas added to the carboxy-terminal primer. The full-length Flagellin XcDNA sequence (SEQ ID NO:75) was expressed as a pET17b (Novagen,Madison, Wis.) construct and resulting recombinant protein was purifiedwith a Ni-NTA affinity column (Qiagen, Valencia, Calif.). The fulllength amino acid sequence of Flagellin X is represented in SEQ IDNO:79. Both Western blot analysis and ELISA assays demonstrated thatthis recombinant flagellin protein was reactive with antibody from micewith IBD.

[0409] Three truncated forms of Flagellin X were constructed and used inexpression studies. The following truncations were cloned using PCRprimers developed from the flagellin X sequence:

[0410] i. the amino terminal conserved end of the molecule (cDNAsequence:SEQ ID NO:76, amino acid sequence:SEQ ID NO:80)

[0411] ii. the amino terminal conserved end plus the variable portion ofthe molecule (cDNA sequence:SEQ ID NO:77, amino acid sequence:SEQ IDNO:81)

[0412] iii. and the carboxy-terminal conserved portion (cDNAsequence:SEQ ID NO:78, amino acid sequence:SEQ ID NO:82)

[0413] A six histidine tag and cloning sites were added to allconstructs for cloning and expression with the pET17b vector. Theconstructs were expressed in E. coli and recombinant protein analyzed byWestern blot and ELISA. Western blot and ELISA assays with aminoterminal and carboxy terminal recombinant protein demonstrated that thegreatest antibody reactivity was to the conserved amino terminus. Theserecombinant proteins have utility in the diagnosis, therapy, and vaccinedevelopment for IBD.

Example 3 IDENTIFICATION OF THE FULL-LENGTH NUCLEOTIDE AND POLYPEPTIDESEQUENCE OF H ELICOBACTER BILIS FLAGELLIN B

[0414] This example describes the identification of the full-length H.bilis flagellin B nucleotide and polypeptide sequence. The recombinantDNA and protein sequences have utility, for example, in the diagnosis,therapy, and vaccine development for IBD.

[0415] Infection with Helicobacter spp, including H. bilis, has beenreported to cause IBD in immunodeficient mice. Thus Helicobacter spp andspecific Helicobacter proteins are useful tools for investigatingmicrobial-induced IBD. Helicobacter spp, especially H. pylori, have alsobeen implicated in human IBD. As shown in Examples 1 and 2 herein,bacterial flagellin are believed to be associated with IBD in the mousemodel. Described herein are the nucleotide and polypeptide sequences forH. bilis flagellin B (FlaB). This sequence was amplified using standardtechniques from H. bilis total genomic DNA using oligonucleotide primersderived from the H. mustelae flagellin B sequence. The full-lengthnucleotide sequence (coding sequence) of the H. bilis flagellin B is setforth in SEQ ID NO:83. The amino acid sequence encoded by thisnucleotide is set forth in SEQ ID NO:84. This polypeptide showedhomology to H. mustelae and H. pylori flagellin B proteins. The aminoterminal conserved region of the H. bilis flagillin B protein includesamino acid residues 1 to 154 of SEQ ID NO:84, and the correspondingnucleotides that encode this region from SEQ ID NO:83. Thecarboxy-terminal conserved portion includes amino acid residues 365 to514 of SEQ ID NO:84, and the corresponding nucleotides that encode thisregion from SEQ ID NO:83. In summary, these sequences representattractive therapeutic and diagnostic targets for IBD.

Example 4 IDENTIFICATION OF THE FULL-LENGTH NUCLEOTIDE AND PROTEINSEQUENCE OF CBIR-1 FLAGELLIN

[0416] This example describes the full-length sequencing of the Cbir-1flagellin clone (partial sequence set forth in SEQ ID NO:1). This clonewas originally obtained by serologic expression screening a mouse cecalbacterium library with serum from mice with IBD (see Example 1 and Table2). The recombinant DNA and protein sequences have utility, for example,in the diagnosis, therapy, and vaccine development for IBD.

[0417] The Cbir-1 clone is a partial bacterial flagellin sequence (aminoend plus variable region) that was shown to be highly reactive with themouse serum used to screen the bacterial library. Recombinant proteinderived from this clone and a recombinant representing the aminoterminus of this cloned sequence were also highly reactive with diseasedmouse (IgG2a) and human serum and also potentially contain a T cellepitope. These data indicate that this protein is immunogenic in mouseand in human. Described herein are the full-length CBir-1 flagellinnucleotide (SEQ ID NO:85) and protein (SEQ ID NO:86) sequences. Thenucleotide sequence was obtained by standard PCR amplificationtechniques from total genomic mouse cecal bacterial DNA with a primerspecific for Clone CBir-1 sequence and a second primer derived from theconserved carboxy end of related flagellin sequences. Polypeptidealignments confirmed that the protein is related to other flagellinproteins. The amino terminal conserved region of the CBir-1 proteinincludes amino acid residues 1 to 147 of SEQ ID NO:86, and thecorresponding nucleotides that encode this region from SEQ ID NO:85. Theamino terminal conserved end plus the variable portion of the moleculeincludes amino acid residues 1 to 418 of SEQ ID NO:86 and thecorresponding nucleotides that encode this region from SEQ ID NO:85. Thecarboxy-terminal conserved portion includes amino acid residues 361 to493 of SEQ ID NO:86, and the corresponding nucleotides that encode thisregion from SEQ ID NO:85. Thus, in summary, these sequences representattractive therapeutic and diagnostic targets for IBD.

[0418]FIG. 1 shows a schematic of flagellin clones with percentsimilarity to related flagellin B from the rumen anaerobe, Butyrivibriofibrisolvens. (A) Structure of B. fibrisolvens flagellin A showingconserved amino and carboxyl regions and the hypervariable centraldomain. (B) Mapping of the predicted amino acid sequences from theflagellin expression clones (1-12) in relation to the B. fibrisolvenssequence. Percentage range (41-84%) indicates the similarity inNH₂-conserved sequence between the clone and B. fibrisolvens sequences.(C) Diagram of the full-length amino acid sequence of mouse cecalbacteria flagellins cBir-1 and Fla^(X) indicating the similarity of thethree domains with the respective B. fibrisolvens domains. (D, E)Schematics of recombinant flagellin proteins and fragments for cBir-1(D) and Fla^(X) (E) that have been expressed and purified in E. coli by6-histidine tag affinity to NiNTA columns (Qiagen Corp.).

Example 5 PEPTIDE PRIMING OF T-HELPER LINES

[0419] Generation of CD4⁺ T helper lines and identification of peptideepitopes derived from bacterial-specific antigens that are capable ofbeing recognized by CD4⁺ T cells in the context of HLA class IImolecules, is carried out as follows:

[0420] Fifteen-mer peptides overlapping by 10 amino acids, derived froma bacterial-specific antigen, are generated using standard procedures.Dendritic cells (DC) are derived from PBMC of a normal donor usingGM-CSF and IL-4 by standard protocols. CD4⁺ T cells are generated fromthe same donor as the DC using MACS beads (Miltenyi Biotec, Auburn,Calif.) and negative selection. DC are pulsed overnight with pools ofthe 15-mer peptides, with each peptide at a final concentration of 0.25μg/ml. Pulsed DC are washed and plated at 1×10⁴ cells/well of 96-wellV-bottom plates and purified CD4⁺ T cells are added at 1×10⁵/well.Cultures are supplemented with 60 ng/ml IL-6 and 10 ng/ml IL-12 andincubated at 37° C. Cultures are restimulated as above on a weekly basisusing DC generated and pulsed as above as antigen presenting cells,supplemented with 5 ng/ml IL-7 and 10 U/ml IL-2. Following 4 in vitrostimulation cycles, resulting CD4⁺ T cell lines (each line correspondingto one well) are tested for specific proliferation and cytokineproduction in response to the stimulating pools of peptide with anirrelevant pool of peptides used as a control.

Example 6 GENERATION OF BACTERIAL-SPECIFIC CTL LINES USING IN VITROWHOLE-GENE PRIMING

[0421] Using in vitro whole-gene priming with bacterial antigen-vacciniainfected DC (see, for example, Yee et al, The Journal of Immunology,157(9):4079-86, 1996), human CTL lines are derived that specificallyrecognize autologous fibroblasts transduced with a specific bacterialantigen, as determined by interferon-γ ELISPOT analysis. Specifically,dendritic cells (DC) are differentiated from monocyte cultures derivedfrom PBMC of normal human donors by growing for five days in RPMI mediumcontaining 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml humanIL-4. Following culture, DC are infected overnight with bacterialantigen-recombinant vaccinia virus at a multiplicity of infection(M.O.I) of five, and matured overnight by the addition of 3 μg/ml CD40ligand. Virus is then inactivated by UV irradiation. CD8+ T cells areisolated using a magnetic bead system, and priming cultures areinitiated using standard culture techniques. Cultures are restimulatedevery 7-10 days using autologous primary fibroblasts retrovirallytransduced with previously identified bacterial antigens. Following fourstimulation cycles, CD8+ T cell lines are identified that specificallyproduce interferon-γ when stimulated with bacterial antigen-transducedautologous fibroblasts. Using a panel of HLA-mismatched B-LCL linestransduced with a vector expressing a bacterial antigen, and measuringinterferon-γ production by the CTL lines in an ELISPOT assay, the HLArestriction of the CTL lines is determined.

Example 7 GENERATION AND CHARACTERIZATION OF ANTI-BACTERIAL ANTIGENMONOCLONAL ANTIBODIES

[0422] Mouse monoclonal antibodies are raised against E. coli derivedbacterial antigen proteins as follows: Mice are immunized with CompleteFreund's Adjuvant (CFA) containing 50 μg recombinant bacterial protein,followed by a subsequent intraperitoneal boost with Incomplete Freund'sAdjuvant (IFA) containing 10 μg recombinant protein. Three days prior toremoval of the spleens, the mice are immunized intravenously withapproximately 50 μg of soluble recombinant protein. The spleen of amouse with a positive titer to the bacterial antigen is removed, and asingle-cell suspension made and used for fusion to SP2/O myeloma cellsto generate B cell hybridomas. The supernatants from the hybrid clonesare tested by ELISA for specificity to recombinant bacterial protein,and epitope mapped using peptides that spanned the entire bacterialprotein sequence. The mAbs are also tested by flow cytometry for theirability to detect bacterial protein on the surface of cells stablytransfected with the cDNA encoding the bacterial protein.

Example 8 SYNTHESIS OF POLYPEPTIDES

[0423] Polypeptides are synthesized on a Perkin Elmer/Applied BiosystemsDivision 430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence is attached to the amino terminus ofthe peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support is carried out using the followingcleavage mixture: trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleaving for 2 hours, the peptides areprecipitated in cold methyl-t-butyl-ether. The peptide pellets are thendissolved in water containing 0.1% trifluoroacetic acid (TFA) andlyophilized prior to purification by C18 reverse phase HPLC. A gradientof 0%-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1%TFA) is used to elute the peptides. Following lyophilization of the purefractions, the peptides are characterized using electrospray or othertypes of mass spectrometry and by amino acid analysis.

Example 9 FLAGELLIN-REACTIVE ANTIBODY IN SERUM FROM COLITIC MICE AND ITSRELATIONSHIP TO PATHOLOGY

[0424] Serum antibody response to recombinant flagellins cBir-1 andFla^(X) and fragments were determined by Western blot analysis. Onehundred micrograms of full length protein (FL) as well as the aminoconserved region (A) and the conserved carboxyl region (C) fragmentswere subjected to SDS-PAGE separation, transfered to membrane andsubjected to serum from non-colitic (pool of 2) and colitic C3H/HeJBir(pool of 5) mice. A second experiment was performed using 20 ngrecombinant protein and serum from non-colitic FVB (pool of 5) andcolitic Mdr1a^(−/−) (pool of 5) mice. Human samples were used in a thirdexperiment. Random human blood donor and a Crohn's patient with severedisease were run against 50 ng of recombinant protein. The results ofthese Western Blot analyses are shown in FIG. 2a. Mouse cecal bacteriaantigens (CBA) was used as a control.

[0425]FIG. 2b shows titration of serum anti-flagellin antibody againstrecombinant flagellins cBir-1 and FlaX with secondary antibodiesspecific for mouse IgG and IgG2a antibodies. Serum from colitic mice(pool of 5) versus non-colitic C3H/HeJBir mice (pool of 2) was used inthe upper panel and serum from colitic MDR mice (pool of 5) versusnon-colitic FVB (pool of 5) mice was used in the lower panel.

[0426]FIG. 2c shows the correlation of colitis score with serumanti-Fla^(X) antibody at a dilution of 1:200 (r=+0.7585). Correlationswere also determined for mouse age versus colitis score (r=+0.3716), andmouse age versus anti-flagellin OD 450 at a 1:200 dilution (r=+0.3253).Colitis scores were based on the following scale: No disease (0-2); milddisease (3-15); moderate disease (16-35); and severe disease (≧36).

[0427] Similar antibody reactivity to recombinant CBir1 and Fla-X wereseen in human Crohn's disease patient sera. As above, anti-flagellinantibodies measured using ELISA in a panel of human serum samples fromabout 150 donors with well characterized human inflammatory boweldiseases, ulcerative colitis and Crohn's Disease, as well as healthycontrols patients (Inflammatory Bowel Disease Center, Cedars-SinaiMedical Center, Los Angles, Calif.). A significantly higher level ofserum anti-flagellin antibody was seen in Crohn's disease patients thanin normal controls, infection controls and ulcerative colitis patients.A significantly higher serum antibody response was seen inpANCA-positive Crohn's disease patients than in pANCA-positiveulcerative colitis patients. These results confirm that the antigensdisclosed herein are useful for subdividing IBD patients into categoriesof disease and would prove useful in clinical diagnosis of IBD,especially Crohn's Disease, FIG. 2d.

Example 10 FLAGELLIN-STIMULATED HUMAN MONOCYTE-DERIVED MACROPHAGESRELEASE INTERLEUKIN 10 AND TUMOR-NECROSIS-FACTOR

[0428] Human monocyte-derived macrophages from seven healthy donors werecultivated in vitro for five days and then stimulated with full-lengthFlagellin X (SEQ ID NO:79) at 6, 12 or 24 μg/ml. Release of cytokinesIL10 and TNF-α was detected in the culture supernatant by ELISA.Cytokine release following stimulation with Flagellin X wasdose-dependent, see FIG. 3.

Example 11 FLAGELLIN PROTEINS IN COMBINATION WITH CHOLERA TOXIN ADJUVANTAS A VACCINE FOR IBD

[0429] Bacterial flagellin protein, FlaX, was used in conjuction with aknown adjuvant, cholera toxin (CT) to prevent the on-set of severecolitis in mice that are genetically prone to the disease. MDR1a mutantmice (MDR1aKO mice) develop severe, chronic inflammation in the colon asthey advance in age. 5-6 week old MDR1a knock out female mice fromTaconic Farms were either left untreated or given 8 μg FlaX or 8 μgMtb+10 μg cholera toxin (List Biologics) in a 200 μl volume of PBS byoral gavage. Mice were thus treated on day 1 and day 14 of theexperiment. Mice were sacrificed 4 weeks after the last dose, and theirlarge intestines (cecum/colon/rectum) were fixed in 10% neutral-bufferedformalin (Sigma), sectioned onto slides, and stained with hematoxylinand eosin (H&E). The sections were examined in a blinded fashion andscored for disease severity.

[0430] Scores ranged from 0 (for no changes seen anywhere) to 64 (mostsevere inflammation and epithelial changes seen through the entire largeintestine) arbitrary units. Typically, mild disease scores as 1-15,moderate disease as 16-35, and severe disease as >35. The mean score foruntreated mice (n=4) was 23.50 with a standard deviation of 17.37. Themean score for mice given control protein plus CT (n=5) was 25.40 with astandard deviation of 18.43. The mean score for mice given FlaX plus CT(n=5) was 6.80 with a standard deviation off 4.38. The mean scores ofthe mice treated with control protein vs treated with FlaX weresignificantly different (Mann-Whitney nonparametric test, p=0.0159).Thus treatment of MDR1aKO mice with FlaX plus CT resulted in less severedisease.

Example 12 IDENTIFICATION OF THE FULL-LENGTH NUCLEOTIDE AND PROTEINSEQUENCE OF CBIR-11 FLAGELLIN

[0431] The full length cDNA sequence of Clone ID 76963, Cbir-11flagellin clone (SEQ ID NO:9) was identified. The nucleotide sequence ofSEQ ID NO:87 is the determined cDNA sequence for clone CBir-11. SEQ IDNO:88 provides a predicted translated flagellin-like sequence proteinsequence encoded by SEQ ID NO:87. A second predicted translated sequenceencoded by SEQ ID NO:87 is provided in SEQ ID NO:89, which is aphosphoesterase-like protein sequence.

Example 13 CREATION OF CBIR-11 FLAGELLIN RESPONSIVE CD4⁺ T CELLS

[0432] CD4⁺ T cells were isolated from mesenteric lymph node cells (MLN)from colitic mdr1a^(−/−), C3 H/HeJBir, C3H/HeJ.IL-10^(−/−) by BD™ IMAGanti-mouse CD4 beads according to manufacturer's instructions (BDBiosciences Pharmingen, San Deigo, Calif.). Briefly, MLN cells werelabeled with anti-CD4 beads ands then placed within the magnetic fieldof the BD Imagnet. The unlabeled cells in suspension were removed andthe cells binding the beads were washed and used in the CD4+ T cellculture. More than 99% of the cells were CD4+ by FACS analysis.

[0433] T cell reactivity to Cbir1 was determined by stimulation assay asfollows, 1.1×10⁵ T cells were incubated in duplicate in the presence of4×10⁵ antigen-pulsed and irradiated APCs (prepared according to Cong etal., J. Exp. Med. 187:855-64, 1998). APCs were treated with 1 to 100μg/ml CBir1 for 18 hours. On day 3 of culture, 0.5 μCi [³H]-thymidinewas added and the cells harvested 16 hours post post. T cells respondedto Cbir1. Non-specific activation via TLR5 or TLR4 activation wasexcluded by the lack of stimulation by Cbir1 or Fla-X onovalbumin-specific proliferation of CD4⁺ T cells from DO11.10ovalbumin-specific TCR transgenic animals.

[0434] A CD4⁺ T cell line reactive to CBir1 was generated by biolatingCD4⁺ T cells from MLN cells from C3H/HeJBir mice as described abovefollowed by culture with splenic antigen presenting cells (APC) thatwere pulsed with CBir1 (100 mg/ml) overnight. The cells wererestimulated every 10-14 days. The T cell line was responsive to CBir1but not to Fla-X or a variety of other microbial, food and epithelialantigens.

[0435] To determine if this T cell line could induce mucosalinflammation, it was adoptively transferred into C3H/HeJ-scid/scidrecipients and quantitative histopathologic scoring performed 8 weekspost-transfer showed that this CBir1-specific CD4⁺ T cell like inducedcolitis in all recipients of an intensity that was similar to or greaterthan that induced by CBA-specific CD4⁺ T cell lines, where as none ofthe recipients given these anti-CD3-activated C3H/HeJBir CD4⁺ T cellsdeveloped disease.

[0436] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1 89 1 1268 DNA Unknown Unknown Bacterium 1 ggaggtatta ttatggtagtacagcacaat ttacatgcaa tgaactctaa cagaatgtta 60 ggcatcacac agaagacagcatctaagtct acagaaaagt tatcttcagg ttacgcaatc 120 aaccgcgcag cagacaacgcagcaggtctt gctatttctg agaagatgag aaagcagatc 180 agaggactta cacaggcttctacaaatgct gaggacggca tcagctctgt acagacagca 240 gaaggcgctt tgacagaagtgcatgatatg cttcagagaa tgaacgagct ggcaattcag 300 gcagcaaacg gcacaaactcagaagatgac cgctcataca ttcaggacga aattgaccag 360 ctgacacagg aaatcgatcgtgttgctgag acaacaaagt tcaatgagac atatctcttg 420 aagggtgaca caaagaacgttgacgctatg gactatacat atagctataa ggcagttaca 480 acgaatactg tagcaagagcttcggtttta gcagcagaga acacagctac aggtatgtca 540 gttagtattt catttgctgcaaacagcggc aaggttactg cagctgactc taacaacctt 600 gcaaaggcta tcagagatcagggcttcaca atcacaacat ctacccagaa tggtaaggtt 660 gtttacggtc ttgagctgaacggaagcgat gcaaaggcaa actatacagt ttcaacagta 720 agtatggaag ctggtacattcaagatcctg aattctaata agcaggttgt tgcatctgta 780 acaatatcta caacagctagctttaaaaag gtatctggta tgtcacagat cgttacggcg 840 tactctgtat cagcagcttatgcgacgggt gatgtatact ctctctatga cgcagacgga 900 aatgcaattt cagcaaacaagctggataag tactttacgg caggcggcgc tacagaggca 960 ggcggaatag ctactacactttcagcaaac tctggtgtgc ctaaggttta tgacgtactc 1020 ggaaaagagg tttctgcagtaagcattgca agtactttag taacagcagt taaggataag 1080 acggctgcat tgaagatgaacttccatgta ggtgctgacg gaacagataa caacaagatt 1140 aagatcaaca ttgaggctatgacagctaag agtcttggag ttaacggtct gaaggtgagc 1200 ggttcgagcg gaacaaatgctacaaatgct atcgagataa tcgctggcgc tatcaagaag 1260 gtttctac 1268 2 336 DNAUnknown Unknown Bacterium 2 gcgcaggaaa aaagttacca gcgtggacaa ggcaaatgtgctggattcct caaggctttg 60 gcggaaagtt gtagaagaag tcggcaaaag agtacccggacgtggcattg gagcatatgc 120 tggtagataa ctgtgccatg cagctagtaa aagacccaaggcagtttgac gtgatcctga 180 cagaaaatat gtttggcgat attttgtccg atgaagcaagcatggtgaca ggctccattg 240 ggatgctttc ctccgccagc ttaaacgata ccaaatttgggctgtatgaa ccaagcggcg 300 gttctgcgcc ggatattgcc gggaaaggga ttgcca 336 3658 DNA Unknown Unknown Bacterium 3 ggcccccgcc ttcggcatgg tgggcaccctggtgggcctg atcaacatgc tgaaggccat 60 ggacatcgag accgttggcg gcaacctgggccccgctatg gccaccgctc tggtcaccac 120 cctctatggt tgcgtgctgg cccacatgatcttcggcccc atcgccaccc agctgcgcca 180 gcgggacgag gaagagaccc tctgcaagctgatcatcgtg gaggggctca tgtccatcca 240 ggccggcgcc aaccccaagt tcctccgggagaagctgctc accttcgtca cccagaaaca 300 gcgtggcgag aacggcggca agaagggcaagtaagagctg cggggccgcg tccccaccgc 360 tctgcggtat gaactgaggt gaaacaccatggcaagcatc aagaaaaaga gctccggcgg 420 cggcggcgcc aactggatgg acacatacggcgacatggtc accctgctgc tgtgtttctt 480 cgtcctgcct gtattccatg tccacgatcgactcggagaa gtggaagatg atcggtccag 540 agcttcaata agaacgcagt cgtcagcgacgatcagcccc ccggaccgga cggcactgaa 600 agcagcacgg gcggcatgaa tctgcctttgacccaggaca tgcaggccgc catggatc 658 4 644 DNA Unknown Unknown Bacterium 4cttgggaagt atttaaaagc gcaggcattg ctggaaaagc tgcgctatta tgcggagaaa 60tgcgggcgca cctatatccg catggaagtg tgcctcctta gcgccgtagc gaaataccga 120acaggcgggg aatggaaggg ggaatttttc ccgatgctga gagaagcctg cggatatcat 180tttatccgcc ttgtcagcga ggaaggggcg gcggcgcagg aactgtttgc ggcggcggga 240aagagtcttc tggaaaagga agtaatggat aaggcatggc tgtccaggct catggaggaa 300acagggaagg tggcggtgcg ctacctggcg tatttaaaag gccggcttgc cgaagcgccg 360gatttctgtg aggcggcatt atccatcctg cgcctgcagg cggaaggaaa gagcgcatca 420atgaagcttg gagttttttt tgttcggcta ttttttggcg tcggtaagct ttacgttttt 480gaaagtcagc acagagaaag gagaggtagg cagcggagta taagacatgc ggaagcaaat 540gaaccaaatg gaggtagaaa catgacgtta tttcaatgaa cagttgttgg aagcaggtgt 600tctttcggac atcagacaag aagatggaac cctaagatgg ctcc 644 5 685 DNA UnknownUnknown Bacterium 5 aatgaaactt atcaaaaaca atctctccat cacacaaatgcttgttgaaa catcatctca 60 tcttgatagc aacactaaaa atattgccaa aatttcacaggataacaccg agctaggcga 120 aaggagtgtg aatatcattg agcaaaacat caccctttcaaatgcaacaa aagaatcttt 180 agaagatgtg cttaatacga tgcagcaaac tcaagcactcataagctcta tcaacgaaga 240 aatcacaaaa gacgcacaaa aagaagatga aaatatgcaaaagattctct ctcttgctaa 300 tgaggcaaaa aatattcaaa gtgtacttgt aactattaccgacatagcag accaaacaaa 360 cctcttagca cttaatgcag ctattgaagc agcgagagctggggagcacg gacgaggctt 420 tgctgtggtg gctgatgaag tgagaaaact agcggagcgcacacagcatt ccattacccg 480 agacaggtag cattatccaa tctgtcttgc agtctattgatgaagtatca agtgatatgg 540 gaaaccagtg ccaaatcaat gaataatctt tcaagcagggtgaagtgatg ttggcaatat 600 acaatctctt gcccactcgt gcaagaaacc aatgcaaaatcattgcaaag gctagaggga 660 gccccaatgg cgaatgaaaa tacca 685 6 929 DNAUnknown Unknown Bacterium 6 atcatttcta cgaaccaggg tgtcatcaca gacaaagaagcaagaaagct cggcgtaggc 60 ggagaagtac tggcatttgt gtggtaggca ctggcactgaacacttagcg aagccaagcg 120 ttccgcgcag gctgaggtta gtgtgaaggc cgttctcagagcgaagcgaa gagaacttcc 180 ttgctaggga acacttagcg aagccaagcg ttccgcgtaggctgaggtta gtgtgaaggc 240 cgttctcaga gcgaagcgaa gagaacttcc ttgctagggaacacttagcg aagccaagcg 300 ttccgcgcag gctgaggttg gtgtgaaggc cgttctcagagcgaagcgaa gagaacttcc 360 ttagaggaaa caccatcgtc actagactcg aaagaaactcgccaagtgat cacagcaact 420 gaaaacagag caggcgaaag gttctgctcc gagaatttaagttaaggagg atatggcaat 480 gtcacgtatc ggaagactgc caatcgcgat tccggcaggagtaactgtgg aaatcgcaga 540 gaataatgta gtgaccgtaa aaggtccaaa gggaactctgtctcgggagc ttcctgttga 600 aatggaaatt aagaaagacg gcgagacaat cgtcgttacaagaccgaatg atttgaagaa 660 gatgaaatcc cttcacggcc ttaccagaac actgattaacaacatggtta tcggcgttac 720 agaaggatat aagaaagttc ttgaagtaaa cggtgttggttatagagcag caaaatcagg 780 aaacaaatta acacttagcc ttggatattc ccatccggtagagatgatcg acccggaagg 840 cgttgagacg gttctcgagg gacagaacaa gattaccgttcagggtatcg acaaggaaaa 900 ggttggacag tatgcagccg agatcagag 929 7 652 DNAUnknown Unknown Bacterium 7 gcatcaaaag catcgctaat tgccgctcct gctggaaccatagggaaaac tttatcatct 60 tcttcaatca cgcattcaat tactactggt ttctttaatgcaatggcttt ctcaatcgca 120 ggcgcaacct cctcacgctt ggtaacccgg attgcctcacagcctaaacc ctctgaaacc 180 tttacaaaat cgaccttatc cgtaagaatg gtctgtgaataccgtttacc ataaaataaa 240 gtctgccact ggcgcaccat acccaaaaca tgattatttatcacaatctg gataattgga 300 atattatagc ggcttgcagt tgccaactca ttcaaattcatccgaaaaca cccgtcacct 360 gcaatattca cacatatttt atctggtctt ccaacctttgcaccgataca tgctccaaga 420 ccatatccca ttgtaccaag acctcccgaa gttaagaaagtacggggttc tgtataccta 480 taaaactgtg ctgcccacat ttgatgctgg cctacccgctcaatttttgg ttgcctatgt 540 agctcagggt agaagcactt cttggtaagg gaagaagtcgcggggttcaa aatcccgcca 600 tcggctttta agtaaaaaac ccccgggagg ggcttatttgcattaatccc cg 652 8 497 DNA Unknown Unknown Bacterium 8 cttcgctcggatggcttcat cctcgaccgc gtcacaaatg ggccctggtg gtctactact 60 gctggttctgctacggacgg tcacctcttg aatacgtacc cgacgaatat ctcccctcag 120 gataaccgttcccgcgggtt cggttttgcc gttcgctgtg tggtacggga ggggtggagg 180 ctaaatctgcttcctacgcg tcgctgggcg tttgcgtggc ggtacggcat ttttcttagc 240 agccccgcgcgtagccggac tagacactcc cgttgcgggt ttccctgcgc ccccccgtgt 300 tgttcgagcggtttttgtag ctcctcttgc tgccgctcta ctattgcttc tcgtagccgt 360 acttctagttgtttttctac ttgatgccct agcgctttcg gcgatttgct tctcgattcg 420 tgcagtaatgctagcgagcg cctcggtgtc acccattgcc tcataaatcg ccaaaatttg 480 ccgcaaagtatttgtac 497 9 327 DNA Unknown Unknown Bacterium 9 gtgtgtggtc cacggcgcggccttcaccgg cggtgagcgc acggaccagg tgctggcgga 60 cttcaccgcc ccggaggacggtcttctcca catcctctgc ctccacggcg acgtcttcag 120 ccaggacagc gtctacggccccatcacccg gccccagatc gcccgcagcg gcgcggatta 180 cctggccctg ggccacgtccaccagtgcag cggcatccag cgccaggggg acaccccctg 240 ggcctacccc ggctgtcccgagggccgggg gttcgacgag ctgggggaca agggtgtgct 300 ggcggggacg gtggatcggggcggggc 327 10 621 DNA Unknown Unknown Bacterium 10 attgagataccattggccga gtgattgcgg cttataggga taaacaaaaa cgtgtctgag 60 atgttacttataaagtgctg ttgtgcctgc aggtgtcatt tctcgaacct ttattgttaa 120 atataataaataaggaaaaa tttctttaat aatacgtcat ataccgcaaa attttagtaa 180 ctttgcactcgggaaaaaac gtgtttttta gaaatcaaaa tttatcaact cccaataaat 240 aacaaggtcatttactacaa tgagcacaat tgaagaaatc aagaaagccc gtgtagccga 300 catcaagaagaatcttgaaa gctacggcat cgacggcaca actgaaatcg tgtacaaccc 360 tacctatgagcagctctttg ctgaagagac actccccttg ctcgaaggat atgaaaaggg 420 tgttgccactgagcttgacg ctgtcaatgt tatgaccggc gtatataccg gccgttcacc 480 caaggacaaattcatcgtac tcgacgaaaa ctcaaaggac accgtatggt gggacaaccg 540 aagaatacaagaacgacaac aagcccgctt ccgaaagagg catggaaggc ttgcaaggaa 600 cttgcagtgaaaggaacctc a 621 11 625 DNA Unknown Unknown Bacterium 11 acggtacgggcaagcgggat cgcgacttta ggatctgcaa gtcccaaagc ctttgccgct 60 ttcagcgcgtcctttcctac agactgtgcg gctgtagcag gacggctacg catcatctca 120 atttcgttatgaatctgagc gcgctgctcg ccggcgcgta cggtcagttg ggcaagtctg 180 cgcgcgacttcgtcatttgc ctgcattatc ggcctccttt gtctgaaccg aaggaaatag 240 agcagataaaaaaagaaatg agggaagcgg gcattaaata aaagaatgtg accgattcgg 300 acaccgaaaaaacaagaaag agaggattta aaaatggcag gatttgacct taatagtttg 360 ctgaatggaaagagcaaagg ggcagcagga cagaagcagg aaacggcggt agcagggcag 420 gggccggcagaggggcagga aagcagtttt gaggttgtaa tgcttgatgt agaggactta 480 atgccaagcaaggataattt ctacacaacc gagggaataa acgagttggc ggacgctatc 540 gagttgtcgggcggtatcga gcagaattta attgtaaagc cgggaagcac acggaaagta 600 tgaggttatcgcaggacacc gcgga 625 12 542 DNA Unknown Unknown Bacterium 12 gatcgctgcctgtttgactc ctgatattgg tcttcaacgt gagcttctct ttgtgcctgc 60 acgaggaggactaggtgaac ggatggagat ccaggcaaat aacgtgtgtg ctcggatggc 120 tcgacggactggtgggaaaa gccgttcgct ctatgtgccc gagcaagtaa gcgagagtac 180 gtatcgtccattgttaaaag aacctgctgt tcaagaggtc gtcaatttga tcggtcaaag 240 taatgctgtgatccacagca tcggaacagc aatgcatatg gcacatcgac gttcgatggc 300 gccagaagtgatcgcaatgc taaataagaa aaaagcggtc ggtgaagcat ttggttactt 360 ttttgatgaaaaagggcaga tcgtgtatcg gatctcacgt atcgggatcc agttagaaga 420 cctccttctatggaatgtgt aattgcccgt cgctggtggc acttcaaaag ctaaagcgat 480 ccgtctcttacatgaaacac ccttctaaac aaacattgtc tgatcacaga ccaaaggaac 540 ca 542 13 585DNA Unknown Unknown Bacterium 13 gggctgcgga ttattaaacc ggggagatataagagtggat gccgaaaaat ggaatcttta 60 atttccggtc atagatgaaa aaaattgaatatagaaatag aagatgttaa gggtgacgaa 120 aggattcgtc gagaactatg acactgtcatagtcagccaa cacaaggagg taattttatg 180 gtagtacagc acaatcttac agcaatgaactcaaacagaa tgttaggaat cacaacagga 240 agtttagcaa aatcagcaga aaaactgtcttcaggatata aggtaaaccg cgcagcagat 300 gatgcagcag gacttgcaat ttcagaaaagatgagaaagc agattagagg tcttacacag 360 gcttcaacca acgcagaaga tggtattagcgcagtacaga cggctgaggg tgcattgact 420 gaagttcatg atatgttaca acgtatgaatgagttggcag taaaagctgc aaacggtcaa 480 tgtctctttc tgacagacag accattccaagatgaagtga cacagcttct cacagaagtt 540 gcccgtgtag cagaaacttc caaatttcaccgaaatttat tcttg 585 14 916 DNA Unknown Unknown Bacterium 14 gtagaagatggaacgtttaa gaccattcac atcacattaa aatatggtgc agataagaac 60 gaaaaagtaatttccggcct taagagaatc tccaaaccgg gtctccgcgt atacgcaaac 120 agcgaagagatgccgaaggt actgggcgga ctcggaattg caatcgtatc tacaaataaa 180 ggtgttgttaccgacaaaga agcaagaaag ctcggcgtag gcggagaagt tctttgcttt 240 gtgtggtgaccggaagcact tggtgaatcc aagcgtaagc gagagatgaa cttagtgcga 300 ggtcgttctttgagcttagc gatgcattga atacttggcg aggtattatc gagcctagta 360 tgaaagcagatattccgaac gaagagagga atatcttcga cagagcttag cgaaaagaac 420 agataaactgaaaacagagc agactcaagc ctctgctccg aaaatttaag ttaggaggac 480 aaaggtatgtcacgtatagg aagactgccg atcgcagtcc ctgcaggtgt aactgtagag 540 attgctgagcataatgtagt gaccgtaaaa ggtccgaagg gaactctcgt aagagaactc 600 ccggttgaaatggaaatcaa gcaggaaggc gaagaaattg ttgtcaccag gccaaacgac 660 ttaaagaggatgaaatccct tcacggcctg acacgtacac tgatcaacaa catggtaatc 720 ggtgtcagccagggttatga aaaggttctg gaagtaaacg gtgttggtta cagagcagcg 780 aagtccggcaacaagctgac cctcagcctt ggatattcac atcctgttga gatggttgat 840 ccggaagggatcgagacagt tctggaaggc cagaacaaga tcacggtaaa agggatcgac 900 aaagaaaaagttggcc 916 15 625 DNA Unknown Unknown Bacterium 15 aatgaatgca aacagaaacttggggatgac cacaaccgca caggcaaaat ctacggagaa 60 gctttcttct ggttaccggatcaaccgcgc ggcggacgat gcggcgggcc tttcgatttc 120 cgagaagatg cgcagccagatccgcggcct gaagcaggct tccaccaatg cgcaggacgg 180 catttccctg attcagactgcagagggtgc gttaaatgag cagcattcga ttttacagag 240 aatgcgcgag ctgtccgttcaggcggcaaa cggtgtggag acggacgacg accgtgaggc 300 agtcaacaac gagatcagccagctccagtc cgagctgaca aggatttccg agacgaccga 360 gttcaacacg atgaagctgctcgacggaag cctttccggg acagccggat cgtccaccgg 420 ctcaggcccg aagttcggcgtggtagatgc aaccttagac ggcgcgcttg tgacgtcgaa 480 tgttgcaggt gtcaaggtagctacagcttg ctacgacaag tacaaaagcc ggacagggag 540 actgccatct gggacgcaacccggaaagac cttgacattg aatctttctc gtgcccgaat 600 tcaaaaaagc tttttcgaagaagta 625 16 603 DNA Unknown Unknown Bacterium 16 aggccatctg aaaatcggacacaatgtgaa aatcggatat ttcgcccaga atcaggctca 60 gcttctcgac ggcgaacttaccgtttttga caccatcgac agggtggctg tcggagatgt 120 ccgcacaaaa atacgcgacatcctcggcgc attcatgttt ggcggagagg cgtcggacaa 180 aaaggtgaag gtgctctcggggggcgaaaa gactcgcctt gcgatgataa aacttctgct 240 tgagccggtg aacctgcttattttggacga accgaccaac cacctcgaca tgaagacaaa 300 ggatatactc aagcaagcgataaaggactt caacggcacg gtgatagttg taagccacga 360 ccgcgaattt cttgacggacttgttgaaaa ggtatatgag ttccggtggt ggggctgtgc 420 gtgaaaacct tggcggaatctacgatttcc ttgaacgcaa gcgccttgct tcattgacgg 480 agttggagcc gaaatgcaccccgggccaaa gatgacaaaa ctcctccccg gcgaagcgag 540 aacactcagc ccgcccacggactcgcaacc tttaagctac cgccgaggcc cgcgaacccc 600 gac 603 17 548 DNAUnknown Unknown Bacterium 17 gtctatatga ttattcaaca taatatagcg gcgattaactcttatcgtaa cttaggcgta 60 aaccagagcg gactgaacaa aaacttagag aaactgtcatctggttacaa aatcaaccgt 120 gcaggcgatg atgcagcagg tctggctatt tccgagagcatgcgttctca gattaacggc 180 ttaaaccagg gcggactgaa caaaaactta gagaaactgtcatctggtta caaaatcaac 240 cgtgcaggcg atgatgcagc aggtctggct atttccgagagcatgcgttc tcagattaac 300 ggcttaaacc aggcagtaaa caacgcaaag gatgccatcggtttgattca gacggcagaa 360 ggtgctctga ccagggattc ggcgtatccg tttccagcggtatgctgcag gattccatcg 420 ctgatacgaa acggattgaa tacacaagag ttgaataaatgaatagagca gcgttgacgg 480 gttgtgcccg tcaacgctgc tttgtccata ggcagttcgattttgccggt acgcattgcc 540 aaataaac 548 18 535 DNA Unknown UnknownBacterium 18 agctacgttg agtaccagac tgcaaatcgt cactatgcac acgttgactgcccgggccac 60 gccgactatg tgaagaacat ggtaactggt gcagctcaga tggacggtgcaatccttgtt 120 tgtgctgcaa ctgacggtcc tatgcctcag acacgtgagc acatcctcctcgcccgtcag 180 gtgaacgttc caagaatcgt tgttttcatg aacaaggttg accttgttgacgatcctgag 240 atgcttgacc tcgtagagat ggagctccgt gacctccttt cattctacaacttcgacggt 300 gacaatgctc cggtaatccg tggctctgca cttggtgcac tcaatggtgaccctcagtgg 360 gaggataagg ttatggaact catggcagct gttgacgagt atattccgcttcctccacgt 420 gacaacgaga agccattcct tatgccaatc gaggacatct tctctatcacagggcgtggt 480 actgtagcaa ctggacgtat cgagacccgg gatcattcac cgtaggtgatgaagg 535 19 505 DNA Unknown Unknown Bacterium 19 aggaaatcca atggtgccgggactgggtat ctcagtatga gaattacgcc atgtatcgga 60 aatacgggct gggagcggtaattctgaaag agggggagcc ggtgtccggc gcgtcctcct 120 ataccggtta tatcggcggcattgagatcg aaattgatac cagagaggac tgccgcagaa 180 agggcctggc ctatatatgcgccgccagac tgattttgga atgtctggac cggggctggt 240 atccaagctg ggacgcgcaaaatctgtggt cggtggcgct ggccgggaag ctgggatatc 300 attttgaccg ggaatatacggcatatatgc gggtcaggta ggggaaaggg aacgtaaaat 360 caagtaccgc gagatttgagagaacataaa aaataacagg aggttccgat agatgaaagt 420 tttaatgctt gaacggcagcccccgtgcca acgggaatac atacgcgctt ctgccggtga 480 aaatggaaaa aaatttttgaacagc 505 20 532 DNA Unknown Unknown Bacterium 20 gtaaggtaat tctgttaaaattgactgggg cgtgtttgga atgttttttg gcagatacca 60 aacgcgccct aagcaagacaggggaatgga atatgagcgt acaggataaa atagaacatg 120 tattaaaatg catacacctgctgttttcca aaagccagcc ttacggggac agcaatacaa 180 agattattgt ggataaaaaagccgtttttg aactgctgga acagttaaac cttgcagttt 240 atgaggctat ggaccattatgaggttacca cccgtaagca tgagattgca gagcggcgct 300 gcgagaaacg gggcgaggaaatcatccaga aggccagcaa gcacgcggac gacatctatg 360 ccgcttccat tatgtataccgacgatgcca tcaaccggat ttgctatatt atggatgacg 420 cccatcaggc tgtccaaaatattttccgta agatgaacgt agagatggaa aaacccctcg 480 tgccgaattc ggccgaaggagtggggttgg taatcgacgc aaagccgacc ca 532 21 467 DNA Unknown UnknownBacterium 21 cttacacaag agcctgccga aatcttaaat cacacctact aatatacaatgcgtgaggat 60 attgtgatgg caatggagga actggagctt acgccgcagc aggcaaaggcactcttgaaa 120 tctccctgtc cgcttgatga tgtgtataag gaatttaagg acagagaggtcgagcatatg 180 gatacgattc gtgattccat tgaaacgaga gcagatcagg ttatcaagcgggaaaacgca 240 agggaaagca ggtgatccta tgccgtatat cccaccggag gtgattgaacaggcaaggca 300 aattgatttg ctctcatata tgaaagcctt tgagccgaat gagctggtcaggatttccgg 360 caacaactac accacccgca ctcacgacag cttaaagatt tcaaacggtaaatggatgtg 420 gtggtcgcag cgaatcggcg ggtataacgc ccttgattat ctcataa 46722 701 DNA Unknown Unknown Bacterium 22 cttttggcac tttttttgat gcaacataaaatttttatcc aggagaatgc ttaatgaaaa 60 accaaaaaca aaccataaaa tgctcagaatgtaaccattg cagcggcttg cgtcgtcccg 120 gcaacacaag cacgagcttc acctgctcccaccctgacca ggggtatatc caggactatt 180 tccgtgaaaa aaggatgaac aagatgcctggtttccttgg atacggggca agatattcgg 240 aggccgtccc tatcaagaca gctcccgcctggtgccctga aaaaagggcg gcaaaacaaa 300 aacgaaacgg gcaatgaccg ctttagaaacaaccggggag aggcagttta tgctgtttct 360 cctctttttc attccactct atctgcataaaaaactcaca tacagaatat gaaattgaat 420 tttgcacata attctgttat aataatacacaaataggcga ttgcgaaaca agttcgcaat 480 attaattcca acagggaaaa attcggtcatgtcggatttt aaggaggtgg atatgagtaa 540 tcggatttta agacatgaaa ataaagcagatagggacatc tgcctgcgaa atggcatggt 600 ggtatgtgaa ggtatgcaat cagagggtttagactgcgga tatatttgat gcttgggcag 660 tttggtcagc cgcttattac cagtgataagctgggtaatc c 701 23 68 DNA Unknown Unknown Bacterium 23 catgggggttttccaacacg ccgttttcca ccatgtcctt ggcgcccagc aacttttcct 60 cggcgggc 68 24572 DNA Unknown Unknown Bacterium 24 tgattatcga atataatcat aaatagctgattttagtttt tatcaataag acagcccata 60 tgggcagaaa attaaggagg acgttttaaaatggctaaag ctaaatttga gagaaacaaa 120 ccacattgca atattggaac cattggtcacgttgaccatg gtaaaacaac tttaacagca 180 gctatcacaa aagttttatc tgagagggttgcaggaaacg aagctactga ctttgaaaac 240 attgacaagg ctccagagga aagggaaaggggtatcacaa tctctaccgc acacgttgag 300 tacgagacag acaacaggca ttatgcacacgttgactgcc caggccatgc tgactatgta 360 aagaacatga tcactggcgc tgcacagatggacggcgcta tccttggtag tgggctgcta 420 cagaccgggc ggttatgggc ttcaagacaaaaaagaacct tattcccttt cttgtccccg 480 tccagggtaa ggggccggtt tccctttaattattccggtt ggaattttcc attggaaacc 540 caaaatggtg gaaccattgg gggtttggga cc572 25 442 DNA Unknown Unknown Bacterium 25 cttttatcac gacttccatgggcgaacgtg gtgacgtcac cgtggaggaa gtacggtccc 60 tcctggagaa ccacttctccggtggactgt cgcgcactgt caccgttgag gacatccagc 120 gggcggtgga ggaatattacaaggtaagcc atagcgatct cgtcggcccg gctcgcagcc 180 gcaccatcgt gcatccccgtcatatcgccg tgtacctctg ccgtcagatg cttgatatgc 240 cccagggcga tatcgggaagaagttcaacc gggaccactc gaccgttatc cactccactc 300 gaaccgttga ggcgatgctcgaggacaaca tggaggttca gagcgacgtg gagaggctca 360 tgaagatcat tcgggaatcctaagtgaaaa actgggggat tgtagggttt atcattataa 420 ttatggcggg taccgtttat gg442 26 566 DNA Unknown Unknown Bacterium 26 gagataatac caagaaacggcagggaaacg gaaaacaggg gagctatatg agaatacagc 60 ataatatctc ggcaattaattcccaaagaa atattatgac aacggatcga gcattatcta 120 agaatctgga gaagctaagttccggataca ggattaaccg cgcgggggac gacgcggcgg 180 gccttgccat atcggaggctatgcggaacc agattaccgc gatgaatcag gggcatgagg 240 aatgtgcagg acggaatttccctggtgcag acggtagaag gcgccctgac agaagtccat 300 accatgctca accgcatgaagggcatggcg gtgcaggccg ccaacgggac atacacggaa 360 tcagaacggg ccatgttaaactcagagatg gaagagctaa aggcagagat tacacgtatt 420 ggtgaatcca caacgttcagcggggtaccg ctctttacga acggtgggat taagaggaac 480 gtcacgctga cctcctattacggctgcacg ctggatctat ccagaggcga agtccacgtt 540 aattattccg gcagtgtgggcagagc 566 27 577 DNA Unknown Unknown Bacterium 27 tattcaaaat gtgcagagaagggaagatga aaaatgaaga taaacacaaa tattgcagcg 60 atcaaagcga gtggacacttgaaccggaca gaggataaga tcactacaag cctggagaga 120 ctttcttccg gataccggataaataaggcg gcagatgatg cggcgggaat ggcgatttcc 180 cagaaaatgc atgcgcagattcgcggatta cagcgtgctt ccagaaatgg tgcggatggt 240 atttccttta tccagaccgcagagggcgcc cttatcgaag tagaaaatat gctgcagaga 300 tgccgtgagc tttctgtgcaggcggctaac cagggtgtga ccatgttgga agataaagag 360 gcgatccaga aagagatcgattctctgatg gaagagattg accgtctttc aactgatacg 420 gaatttaaca cgaaaagtattctggatggt tcctgctgtc gtcagacatc ttctaataat 480 attggagtga aggttgtttccatgacagac tcggtgggaa atgacaaaat attggaatgg 540 ctttggacca ggtggccacgaagaccattt tttccat 577 28 361 DNA Unknown Unknown Bacterium 28ggaggggctg aaggaggtgg cccaggggat cgccgcggcg gagcagcggc ggacggtgta 60taccgtggcc cagggggaca ccctgtgggg ggtggccggg cggtacggcg tgaccataga 120ggcgctgctg cgggccaatc cggccatcaa gaaccccaac ctgatccggg tgggacagca 180ggtggtggtg ccggtatgac agtgcggctc ttgacggtgg acggccggca gtttgagctg 240ccggtgaccc tccggtggcg tatcctgcgt accggcggcg tgccctgcga cgagatggag 300gcggtgtgcc tctacgatgg gaagctgggg gcaatcctgc ccctgtgcca ccggttcgcc 360 c361 29 596 DNA Unknown Unknown Bacterium 29 gcagcagaaa gactttcgtccggttacaga gtcaaccgtg cagcagatga tgcggcagca 60 atggcgattt ctgagaaaaaacgggcacag atacgagggc ttgcccgcgc ttcaaaaaat 120 gcacaggatg ggattagttttgtacagact ggagatggag ccatgagcca gattggggca 180 atgctccacc gaatgcgggagctcacggtc caggcgttga atgacggggt ctacgaacca 240 gctgaccggg cggcgctgcagatggagttt gatcagctgc agggtgagat tgaccgggtg 300 aacgaccaga cagaatttaataaaaaacct gtatttgaac attatacaga caatttttca 360 ttgcttgagg gaaaccgggtttggagccag gatcagatcc ataccatcga tagcagcaat 420 tcctctctca cggtgaaatatattgcggtg gagccagacg ggacagaggt agaaaaagaa 480 aagacactta cgattcctgaggggacatat accacccggg aattgatgga tgaaatggat 540 aatgtggtgt cagcgctgggagatgaggcg gatggactgt atctggaata tagtgg 596 30 389 DNA Unknown UnknownBacterium 30 aaccgaagcc ggtctggaag tctatggcca cactctatgc tggcatgaacagcagaccgt 60 gaagtggctg aactctctaa tcaaggataa ggagcttgag gtggatcctaacgagaaggt 120 ggaaaaagag gattatgtta tggattattc tactgtttca tcttacaatttctgggctcc 180 cgatgaagtt aagccaaaca ttacgataaa tgagggttgc tttgaacttgtaaacgcagc 240 tgctactgac aattggaaga tccaatatca tgttgccgac ggacttccgattgaaaaagg 300 aaaatcgtac aggcttaaag tgatggcccg tggtaccggt gaaggtacgatggaaggtaa 360 agtcggtgat tggggaggcg gatcaaatg 389 31 592 DNA UnknownUnknown Bacterium 31 agaaacatgg gtcttcttca tggaagactc gctagacccacactctcttg tgccactgca 60 tgatgactga ttagaatgag agccatggtt agcagcctcgtgccggaaaa caacacttaa 120 cataccttag atttacgcga tcatgaaaac aaaacgacttttactcggtg acgaggcgtt 180 tgcattaggt gcaatcaatg ccggtctgtc aggcgcatatgcatatcccg gcacaccgtc 240 cactgaaata atggagtatg tgcagacaaa tcctgtggcaaaggagcgag gtatacattc 300 ccattggtca tcaaacgaga agacagcaat ggaagaagctctcggcatgt cattctgcgg 360 taagcgtaca ttcacgtcaa tgaagcatgt gggtctgaatgtagccgccg atcctttcgt 420 caattctgca atgacgggag cgaacggcgg tcttcttgtcgtggcggcag atgatcccgg 480 aatgcattcg tcgcaaaatg agcaggattc gcgtttttatgctgattttg cgatgattcc 540 cgcacttgag ccttcagatc agcaaggagg cttatgatatggcgcgtgcg gg 592 32 483 DNA Unknown Unknown Bacterium 32 ggggcttgcgacgaccacgt cgaagtcgaa ccagttctcc ttctggatct tctccaccca 60 ttctgtattggtatcattga gagattgaat cgttaattcg gtcatgcggt gaagaatatc 120 atgtgcttcatttaaagcac catctcccgt ctgtacccag gagatgccat cctcgacatt 180 cttgcttccctggtttagcc ctcgaatcat atagcgcatt ttttcagaaa tggcaagtcc 240 tgcggtatcatcagcagctt tgttgatacg atagccagag gaaagcttct cgacagactt 300 ttgtttcttattggtgttga tattcagttg atttgccgta tttaaggcag atagattagt 360 gntgacaattaacatattta gcccctcttt ctaaaataat catgttattg caaatccatc 420 gccaatattataaatttttg agtttcccca ttttgcacat aaaagcgcga tggggtcgct 480 ccc 483 33214 DNA Unknown Unknown Bacterium 33 gcttcaagaa aattttataa attttttaagaaaaaataga tttgcccaca atgcccacgg 60 agtatgtggt attatggtat tgtcgaaagacaagggaaga gaggtattta gatactcctt 120 ttataagcac tcaaactggc gatagaaaaagcatctcaaa agggtgcttt ttctgttgta 180 gagaaacagg tgggaaggtg gtgtcgcggctcat 214 34 375 DNA Unknown Unknown Bacterium 34 tatacggaga gcttgtccaggcttttacgg tttgccggaa accgcatgga agcggtgggg 60 ctataccaga tgatacttcaggttttgtgg ctgattctgc tgtttgcagg gatcagcctg 120 ctctttggcc gcgtggcaggaatcgtgacc ggaagtgttc tggcggtttc tttctggatg 180 atggagacca ttcttgtaatttggcccgga aacttctata tgctgcattt tactatagcg 240 cttatgttct taggatacgtcagataccgg atcaaaaaag ggggatggcc ctcaaacgat 300 tttggccggc tgtgcctggctgccatcggt ttttatgtgg gcgttctctg catttgggat 360 attctggggg gcata 375 35481 DNA Unknown Unknown Bacterium 35 attgcaatcg tgatgaaccg tacgtgtggtaagctcacgt ttcgaagatg ttgtgtatta 60 ctctattctg ccacttaaga ttctcaccgatacaagcacg ggcacaaatc tctttgaagt 120 ctatacaaaa agccttaaag aagggattacaggcactcaa gaatcgttgc aaaatgttct 180 taaagaatct ggcgaagtag cagaatattctacaaatgcg gcacaatcag ctgatgatgg 240 cttagatatg agtaaaaagg cattagaagaaattgaagtt ttgtatgaaa aaatgcaaat 300 cgcttctgat ttggtggatt cactcacacaaagaagcaat gaaattacaa gcgtaatctc 360 gcttattgat gatattgcgg aacaaacaaacctccttgcg ctcaatgcag ctattgaagc 420 accgagagct ggggagcacg gacgaggctttgccgtgggc gctgatgagg tgcgaaaact 480 c 481 36 503 DNA Unknown UnknownBacterium 36 agctgttgat ggcgacatgg acaaagctat tgatttcttg cgtgaaaaaggtatggcaaa 60 ggctgctaag aagagcgacc gtgttgctgc tgaaggttta gctgatgttgaagttgtagg 120 gaacatagct gcagttgttg aaatcaacgc tgaaacagac tttgttgctcaaaaccaaca 180 attcaaggac cttgtaaaac gtgttgcagg tttgatcgca gaaaataaaccagctgactt 240 agaagctgct ttggctatca agactgacaa aggtacgatc aacgaagaaatcatcgaagc 300 tacacaagtt atcggtgaaa agatcacatt gcgtcgtttt gaattagttgaaaaagctga 360 caatgaaaac ttcggtgctt acctacacat gggtggtaag atcgccgttttaactgtggt 420 tgaaggtgct gatgaagtgg ctgctaaaga cgttgcaatg cacgttgcagctatcaatcc 480 taagtacgtg aaccgcgacc aag 503 37 587 DNA Unknown UnknownBacterium 37 cagtgtttct ccggataatc cctttttccc attctctatt tcggataaaaaatttactga 60 aatgtcaacg ctttccgcaa attcggcctg cgtaaaatca ttcaggatccgcagctgccg 120 aatccgttgc ccaatttcgg ttttgttaag agtatctttc ataggttcctccagaaaagc 180 ggctgcttgt aacagtgggg aaactacagg actgttacgg ccaaattgccatgattatgg 240 aatgtttgtg ttgctattag tttattttac caatccttaa aaaatagaaaacaaataaaa 300 gattgaatta attctctatt agcgataaaa tataaactaa aagtgacgatatacataata 360 agactggata gggaggagtg gttggaatgg tgattgcaaa taatctattgtcccagttca 420 cagcaaggca gttaaatata aatagcggta agaaagaaaa agcggcagagaaactttctt 480 ccggctaccg cattaacagg gcgtcagata atgcggcggg cttaaaaatttcggaaaaaa 540 tgcgtatgca gatccgcggg cttatgccgg ggcgcacaaa ataccca 58738 422 PRT Unknown Unknown Bacterium 38 Gly Gly Ile Ile Met Val Val GlnHis Asn Leu His Ala Met Asn Ser 5 10 15 Asn Arg Met Leu Gly Ile Thr GlnLys Thr Ala Ser Lys Ser Thr Glu 20 25 30 Lys Leu Ser Ser Gly Tyr Ala IleAsn Arg Ala Ala Asp Asn Ala Ala 35 40 45 Gly Leu Ala Ile Ser Glu Lys MetArg Lys Gln Ile Arg Gly Leu Thr 50 55 60 Gln Ala Ser Thr Asn Ala Glu AspGly Ile Ser Ser Val Gln Thr Ala 65 70 75 80 Glu Gly Ala Leu Thr Glu ValHis Asp Met Leu Gln Arg Met Asn Glu 85 90 95 Leu Ala Ile Gln Ala Ala AsnGly Thr Asn Ser Glu Asp Asp Arg Ser 100 105 110 Tyr Ile Gln Asp Glu IleAsp Gln Leu Thr Gln Glu Ile Asp Arg Val 115 120 125 Ala Glu Thr Thr LysPhe Asn Glu Thr Tyr Leu Leu Lys Gly Asp Thr 130 135 140 Lys Asn Val AspAla Met Asp Tyr Thr Tyr Ser Tyr Lys Ala Val Thr 145 150 155 160 Thr AsnThr Val Ala Arg Ala Ser Val Leu Ala Ala Glu Asn Thr Ala 165 170 175 ThrGly Met Ser Val Ser Ile Ser Phe Ala Ala Asn Ser Gly Lys Val 180 185 190Thr Ala Ala Asp Ser Asn Asn Leu Ala Lys Ala Ile Arg Asp Gln Gly 195 200205 Phe Thr Ile Thr Thr Ser Thr Gln Asn Gly Lys Val Val Tyr Gly Leu 210215 220 Glu Leu Asn Gly Ser Asp Ala Lys Ala Asn Tyr Thr Val Ser Thr Val225 230 235 240 Ser Met Glu Ala Gly Thr Phe Lys Ile Leu Asn Ser Asn LysGln Val 245 250 255 Val Ala Ser Val Thr Ile Ser Thr Thr Ala Ser Phe LysLys Val Ser 260 265 270 Gly Met Ser Gln Ile Val Thr Ala Tyr Ser Val SerAla Ala Tyr Ala 275 280 285 Thr Gly Asp Val Tyr Ser Leu Tyr Asp Ala AspGly Asn Ala Ile Ser 290 295 300 Ala Asn Lys Leu Asp Lys Tyr Phe Thr AlaGly Gly Ala Thr Glu Ala 305 310 315 320 Gly Gly Ile Ala Thr Thr Leu SerAla Asn Ser Gly Val Pro Lys Val 325 330 335 Tyr Asp Val Leu Gly Lys GluVal Ser Ala Val Ser Ile Ala Ser Thr 340 345 350 Leu Val Thr Ala Val LysAsp Lys Thr Ala Ala Leu Lys Met Asn Phe 355 360 365 His Val Gly Ala AspGly Thr Asp Asn Asn Lys Ile Lys Ile Asn Ile 370 375 380 Glu Ala Met ThrAla Lys Ser Leu Gly Val Asn Gly Leu Lys Val Ser 385 390 395 400 Gly SerSer Gly Thr Asn Ala Thr Asn Ala Ile Glu Ile Ile Ala Gly 405 410 415 AlaIle Lys Lys Val Ser 420 39 73 PRT Unknown Unknown Bacterium 39 Met LeuVal Asp Asn Cys Ala Met Gln Leu Val Lys Asp Pro Arg Gln 5 10 15 Phe AspVal Ile Leu Thr Glu Asn Met Phe Gly Asp Ile Leu Ser Asp 20 25 30 Glu AlaSer Met Val Thr Gly Ser Ile Gly Met Leu Ser Ser Ala Ser 35 40 45 Leu AsnAsp Thr Lys Phe Gly Leu Tyr Glu Pro Ser Gly Gly Ser Ala 50 55 60 Pro AspIle Ala Gly Lys Gly Ile Ala 65 70 40 105 PRT Unknown Unknown Bacterium40 Met Val Gly Thr Leu Val Gly Leu Ile Asn Met Leu Lys Ala Met Asp 5 1015 Ile Glu Thr Val Gly Gly Asn Leu Gly Pro Ala Met Ala Thr Ala Leu 20 2530 Val Thr Thr Leu Tyr Gly Cys Val Leu Ala His Met Ile Phe Gly Pro 35 4045 Ile Ala Thr Gln Leu Arg Gln Arg Asp Glu Glu Glu Thr Leu Cys Lys 50 5560 Leu Ile Ile Val Glu Gly Leu Met Ser Ile Gln Ala Gly Ala Asn Pro 65 7075 80 Lys Phe Leu Arg Glu Lys Leu Leu Thr Phe Val Thr Gln Lys Gln Arg 8590 95 Gly Glu Asn Gly Gly Lys Lys Gly Lys 100 105 41 76 PRT UnknownUnknown Bacterium 41 Met Ala Ser Ile Lys Lys Lys Ser Ser Gly Gly Gly GlyAla Asn Trp 5 10 15 Met Asp Thr Tyr Gly Asp Met Val Thr Leu Leu Leu CysPhe Phe Val 20 25 30 Leu Pro Val Phe His Val His Asp Arg Leu Gly Glu ValGlu Asp Asp 35 40 45 Arg Ser Arg Ala Ser Ile Arg Thr Gln Ser Ser Ala ThrIle Ser Pro 50 55 60 Pro Asp Arg Thr Ala Leu Lys Ala Ala Arg Ala Ala 6570 75 42 152 PRT Unknown Unknown Bacterium 42 Met Ala Met Ser Arg IleGly Arg Leu Pro Ile Ala Ile Pro Ala Gly 5 10 15 Val Thr Val Glu Ile AlaGlu Asn Asn Val Val Thr Val Lys Gly Pro 20 25 30 Lys Gly Thr Leu Ser ArgGlu Leu Pro Val Glu Met Glu Ile Lys Lys 35 40 45 Asp Gly Glu Thr Ile ValVal Thr Arg Pro Asn Asp Leu Lys Lys Met 50 55 60 Lys Ser Leu His Gly LeuThr Arg Thr Leu Ile Asn Asn Met Val Ile 65 70 75 80 Gly Val Thr Glu GlyTyr Lys Lys Val Leu Glu Val Asn Gly Val Gly 85 90 95 Tyr Arg Ala Ala LysSer Gly Asn Lys Leu Thr Leu Ser Leu Gly Tyr 100 105 110 Ser His Pro ValGlu Met Ile Asp Pro Glu Gly Val Glu Thr Val Leu 115 120 125 Glu Gly GlnAsn Lys Ile Thr Val Gln Gly Ile Asp Lys Glu Lys Val 130 135 140 Gly GlnTyr Ala Ala Glu Ile Arg 145 150 43 127 PRT Unknown Unknown Bacterium 43Leu Arg Ser Asp Gly Phe Ile Leu Asp Arg Val Thr Asn Gly Pro Trp 5 10 15Trp Ser Thr Thr Ala Gly Ser Ala Thr Asp Gly His Leu Leu Asn Thr 20 25 30Tyr Pro Thr Asn Ile Ser Pro Gln Asp Asn Arg Ser Arg Gly Phe Gly 35 40 45Phe Ala Val Arg Cys Val Val Arg Glu Gly Trp Arg Leu Asn Leu Leu 50 55 60Pro Thr Arg Arg Trp Ala Phe Ala Trp Arg Tyr Gly Ile Phe Leu Ser 65 70 7580 Ser Pro Ala Arg Ser Arg Thr Arg His Ser Arg Cys Gly Phe Pro Cys 85 9095 Ala Pro Pro Cys Cys Ser Ser Gly Phe Cys Ser Ser Ser Cys Cys Arg 100105 110 Ser Thr Ile Ala Ser Arg Ser Arg Thr Ser Ser Cys Phe Ser Thr 115120 125 44 143 PRT Unknown Unknown Bacterium 44 Met Ser Arg Ile Gly ArgLeu Pro Ile Ala Val Pro Ala Gly Val Thr 5 10 15 Val Glu Ile Ala Glu HisAsn Val Val Thr Val Lys Gly Pro Lys Gly 20 25 30 Thr Leu Val Arg Glu LeuPro Val Glu Met Glu Ile Lys Gln Glu Gly 35 40 45 Glu Glu Ile Val Val ThrArg Pro Asn Asp Leu Lys Arg Met Lys Ser 50 55 60 Leu His Gly Leu Thr ArgThr Leu Ile Asn Asn Met Val Ile Gly Val 65 70 75 80 Ser Gln Gly Tyr GluLys Val Leu Glu Val Asn Gly Val Gly Tyr Arg 85 90 95 Ala Ala Lys Ser GlyAsn Lys Leu Thr Leu Ser Leu Gly Tyr Ser His 100 105 110 Pro Val Glu MetVal Asp Pro Glu Gly Ile Glu Thr Val Leu Glu Gly 115 120 125 Gln Asn LysIle Thr Val Lys Gly Ile Asp Lys Glu Lys Val Gly 130 135 140 45 153 PRTUnknown Unknown Bacterium 45 Val Tyr Met Ile Ile Gln His Asn Ile Ala AlaIle Asn Ser Tyr Arg 5 10 15 Asn Leu Gly Val Asn Gln Ser Gly Leu Asn LysAsn Leu Glu Lys Leu 20 25 30 Ser Ser Gly Tyr Lys Ile Asn Arg Ala Gly AspAsp Ala Ala Gly Leu 35 40 45 Ala Ile Ser Glu Ser Met Arg Ser Gln Ile AsnGly Leu Asn Gln Gly 50 55 60 Gly Leu Asn Lys Asn Leu Glu Lys Leu Ser SerGly Tyr Lys Ile Asn 65 70 75 80 Arg Ala Gly Asp Asp Ala Ala Gly Leu AlaIle Ser Glu Ser Met Arg 85 90 95 Ser Gln Ile Asn Gly Leu Asn Gln Ala ValAsn Asn Ala Lys Asp Ala 100 105 110 Ile Gly Leu Ile Gln Thr Ala Glu GlyAla Leu Thr Arg Asp Ser Ala 115 120 125 Tyr Pro Phe Pro Ala Val Cys CysArg Ile Pro Ser Leu Ile Arg Asn 130 135 140 Gly Leu Asn Thr Gln Glu LeuAsn Lys 145 150 46 110 PRT Unknown Unknown Bacterium 46 Met Met Cys IleArg Asn Leu Arg Thr Glu Arg Ser Ser Ile Trp Ile 5 10 15 Arg Phe Val IlePro Leu Lys Arg Glu Gln Ile Arg Leu Ser Ser Gly 20 25 30 Lys Thr Gln GlyLys Ala Gly Asp Pro Met Pro Tyr Ile Pro Pro Glu 35 40 45 Val Ile Glu GlnAla Arg Gln Ile Asp Leu Leu Ser Tyr Met Lys Ala 50 55 60 Phe Glu Pro AsnGlu Leu Val Arg Ile Ser Gly Asn Asn Tyr Thr Thr 65 70 75 80 Arg Thr HisAsp Ser Leu Lys Ile Ser Asn Gly Lys Trp Met Trp Trp 85 90 95 Ser Gln ArgIle Gly Gly Tyr Asn Ala Leu Asp Tyr Leu Ile 100 105 110 47 22 PRTUnknown Unknown Bacterium 47 Met Gly Val Phe Gln His Ala Val Phe His HisVal Leu Gly Ala Gln 5 10 15 Gln Leu Phe Leu Gly Gly 20 48 140 PRTUnknown Unknown Bacterium 48 Asp Asn Thr Lys Lys Arg Gln Gly Asn Gly LysGln Gly Ser Tyr Met 5 10 15 Arg Ile Gln His Asn Ile Ser Ala Ile Asn SerGln Arg Asn Ile Met 20 25 30 Thr Thr Asp Arg Ala Leu Ser Lys Asn Leu GluLys Leu Ser Ser Gly 35 40 45 Tyr Arg Ile Asn Arg Ala Gly Asp Asp Ala AlaGly Leu Ala Ile Ser 50 55 60 Glu Ala Met Arg Asn Gln Ile Thr Ala Met AsnGln Gly His Glu Glu 65 70 75 80 Cys Ala Gly Arg Asn Phe Pro Gly Ala AspGly Arg Arg Arg Pro Asp 85 90 95 Arg Ser Pro Tyr His Ala Gln Pro His GluGly His Gly Gly Ala Gly 100 105 110 Arg Gln Arg Asp Ile His Gly Ile ArgThr Gly His Val Lys Leu Arg 115 120 125 Asp Gly Arg Ala Lys Gly Arg AspTyr Thr Tyr Trp 130 135 140 49 198 PRT Unknown Unknown Bacterium 49 AlaAla Glu Arg Leu Ser Ser Gly Tyr Arg Val Asn Arg Ala Ala Asp 5 10 15 AspAla Ala Ala Met Ala Ile Ser Glu Lys Lys Arg Ala Gln Ile Arg 20 25 30 GlyLeu Ala Arg Ala Ser Lys Asn Ala Gln Asp Gly Ile Ser Phe Val 35 40 45 GlnThr Gly Asp Gly Ala Met Ser Gln Ile Gly Ala Met Leu His Arg 50 55 60 MetArg Glu Leu Thr Val Gln Ala Leu Asn Asp Gly Val Tyr Glu Pro 65 70 75 80Ala Asp Arg Ala Ala Leu Gln Met Glu Phe Asp Gln Leu Gln Gly Glu 85 90 95Ile Asp Arg Val Asn Asp Gln Thr Glu Phe Asn Lys Lys Pro Val Phe 100 105110 Glu His Tyr Thr Asp Asn Phe Ser Leu Leu Glu Gly Asn Arg Val Trp 115120 125 Ser Gln Asp Gln Ile His Thr Ile Asp Ser Ser Asn Ser Ser Leu Thr130 135 140 Val Lys Tyr Ile Ala Val Glu Pro Asp Gly Thr Glu Val Glu LysGlu 145 150 155 160 Lys Thr Leu Thr Ile Pro Glu Gly Thr Tyr Thr Thr ArgGlu Leu Met 165 170 175 Asp Glu Met Asp Asn Val Val Ser Ala Leu Gly AspGlu Ala Asp Gly 180 185 190 Leu Tyr Leu Glu Tyr Ser 195 50 115 PRTUnknown Unknown Bacterium 50 Met Glu Ala Val Gly Leu Tyr Gln Met Ile LeuGln Val Leu Trp Leu 5 10 15 Ile Leu Leu Phe Ala Gly Ile Ser Leu Leu PheGly Arg Val Ala Gly 20 25 30 Ile Val Thr Gly Ser Val Leu Ala Val Ser PheTrp Met Met Glu Thr 35 40 45 Ile Leu Val Ile Trp Pro Gly Asn Phe Tyr MetLeu His Phe Thr Ile 50 55 60 Ala Leu Met Phe Leu Gly Tyr Val Arg Tyr ArgIle Lys Lys Gly Gly 65 70 75 80 Trp Pro Ser Asn Asp Phe Gly Arg Leu CysLeu Ala Ala Ile Gly Phe 85 90 95 Tyr Val Gly Val Leu Cys Ile Trp Asp IleLeu Gly Gly Ile Pro Arg 100 105 110 Ala Glu Phe 115 51 132 DNA UnknownUnknown Bacterium 51 gttcccatga tctcaaaaca ttctttatat tccatcaggcggcttccgca tttctccagt 60 tcttcataaa gctgctggat ctgcggtttt ttctttcccttaccgcaaac aaactccatt 120 ccttccccta cc 132 52 511 DNA Unknown UnknownBacterium 52 cctggcagag cacgccgcac gccataaccg ggagcggcat cgcattcaaatcctccgcca 60 actggaaata ctccatataa ccaagcccca gcgtcatcat ataaccccagacattaaaat 120 tttccctgcg tttctcgacg atatccaccg aatccttcca atcataaacattatcccaga 180 tataggaacc ttccgagata cagccgccag gaaagcgaag gaaagtaggatgcagctctc 240 tcattgtctc tacaaggtcg cggcgcagac ggtaattcgg attcctaagataatttttat 300 gggcgctcgc gctgccctcc tcctcaccaa atccccagac atgttccgggaacatcgaaa 360 ccatatcgat ggatatactg ccggaaaaag ccaattctaa ctgcccgaaacactccttct 420 ctgccgtaag taccaccgct tgtttttccc cgtatttctt ccagtcgccggatatggaaa 480 ttttttccac attgctgacc gccgcccccg a 511 53 213 DNA UnknownUnknown Bacterium 53 atcgggaaaa agaaatactt tttatggatt tgcgacagataggaagtaca tatgaaaaga 60 gtaccctttt ccgtatttct gcccgcccgg tcggtcagaaatggcttgtt gggaagtgtc 120 ccaaaccctc tatgagaacg gaaaggagcg aaaacccatgaacggacgca aaaggacggn 180 acaggtcaaa ttctatgnga cagangaaga aac 213 54511 DNA Unknown Unknown Bacterium 54 gtacaggttc tcggtctcga ccccgcagtgggccagaagc cgggaggccc cgttgccctt 60 ggtgcgcagc aggcccagga gcagatgctcggagttcacc gcgttctggc ccagccggcg 120 gctctcctcc accgcgccct ggatggctgagcagcagttg ggcgtaagtc cctgaaagct 180 ggactgggcg ggcactcccg tccccacccgctgggcgatg gcggagcgca gggcctggct 240 gtccgccccc gcccggcgca gggccatggcggcggggctg aactcctggc cggccagccc 300 aagcagaaga tgctcgctga atagccaaataaaaccatat cactaatgtt atacaatctt 360 cctgaaaaac agccattact gcatgtaatcaatctccctt ttgcttttgt ttcaatatga 420 agcgggtttt cccacaatat ctgcattaaataagataaat taccctcccc atatattctc 480 tgatcggttc gtgtttttaa tatatattca c511 55 511 DNA Unknown Unknown Bacterium 55 cctacaggcc aggacatgacggcttgcgcg agctttacct gaattgccgc cgccttgtat 60 atgcctacaa taatctccatcctgacaaga ttggggaaag ggacgacctg attcgaagga 120 ttctcggcaa atgcggcgagcgtgtggcag tggagccgcc ctttcattgt gattatggcg 180 ttcatattga agttggcgacaatttttttg ccaacttcaa ttgcgtaatc ctggatgttg 240 ccagggtcat gatcggcaaaaatgtcatgt ttgcgcccaa tgtgggaatt tatgcggctg 300 gccatcctgt tcattggcaaagccgcaatt cgggctatga atatggtcgg gaaatcagga 360 ttggcgacaa tgtctggcttggcgggaatg ttatcgtcaa tcccgggata aatataggca 420 ataatgttgt tgtaggctcgggcagcgttg ttaccaggga tatcccggac aatatgctgg 480 cagcgggtaa tccctgcagggttttgcgcg a 511 56 279 DNA Unknown Unknown Bacterium 56 gataattatgaggaggcctt ggacagtgta gaggaagtca agcgttccct tctggtagcg 60 ttggtggaccgcaaggtgag caaatatttc tccgagcggg acagcattat taagaaaata 120 gaaaaagacaaatattttgt ggcatttaag caaaaatatc tggacgagct gattgaggac 180 aagtttagtatccttgagga cgtcaagact gttaaagtgg gaaatgagat ggcagtcacc 240 ttgagcattggggtgggcgt cagcgggaac agttatacc 279 57 234 DNA Unknown Unknown Bacterium57 cacttacgaa gatgtggtac tgccgcctct atggcatcat aatattacag tatttgatgc 60ctagagcatg tctgaaaatt cattcccgcc atcttcacgc cccactttgc gatttatttg 120gctctcattc ggtcaacgta gctcactacg cctaccacat gatctacttt cgaaaaaaca 180accttaaata accttacatt tcaccagtct atttttctgg cgaaatgtgt ggtc 234 58 164DNA Unknown Unknown Bacterium 58 acattgcgaa atacaatctc acctttcggctggaccaact cctgcgcgtc cggctcatca 60 aaaatctcta tatcagcatc cataatctgcaagaaccgct caatgccagt gattccgcgc 120 tgaaactgct ctgcaaactc aataatcctgcggattgtcg caac 164 59 511 DNA Unknown Unknown Bacterium 59 gagaaaaaattttctttgaa ccggaaaaaa caaaggtcgc cattgtgaca tgcggcggtt 60 tgtgtccggggctcaacgat gtgatccgtt ccattgtcat ggaattatgg catggatacg 120 gcgtgcgggacattatcggc attccttacg gcttggaggg gtttatccct aaaaaatacg 180 gacataagcttattgagctg acccctgatg ctgtttcaac aatttatatg ttgggcggaa 240 caattttaggttcttcacgc ggagcgcaga attttgacga aatagcggat tttttgaatg 300 agaaagggattaatttgctt ttcgtgcttg gcggcgacgg taccatgaaa gccgcaaagg 360 ctatttcaacagcggtgaaa gagcatggtt tgaccctgtc ggntatcggt attcctaaaa 420 ctattgataatgacatcaat tttgtggagc agtctttcgg gttcgccacc gctgttgacg 480 aggcgacaaaagccattgcg gcggcgcata c 511 60 208 DNA Unknown Unknown Bacterium 60actcggcgta ccgggccatg gtatgggggt actggtactg ggagaaggtg cccatcttgg 60gcgggcactc cgcgctgttg tattccacca ccagggagat cagcagtgcg ttggctacgc 120cgtggggcag gtggtggaac gcgcccagat tgngggccat ggagtggcat acncccatca 180accaattngc aaaggccatg ccagccag 208 61 275 DNA Unknown Unknown Bacterium61 agcgtggaaa tcccatccag tgtgaaatgg atattggatt ctgccttttc aagatgcagc 60agcttaacaa gcgtggcaat tcccccaagc gtgacaggga tagcttataa tgctttctta 120ggctgcagcg acaacctcgt aatcgttgga actcctggtt cagaagcaga gaaatatgca 180caacagaaca acattacatt taaatttctt gattccgttc aggacatatc cgaagcatct 240ataaccttgg agagggcaag ctacacctat gacgc 275 62 357 DNA Unknown UnknownBacterium 62 cctgtttctt taaacatggc gtccagccgt ttctgaatat tctcccagatggcatagccg 60 ttgggccgga taatcataca tccctttata ttggaatact ccaccagctccgccttgcgc 120 accacatccg tataccactg cgcgaaatcc tcgttcatcg acgtaatcgcctcaaccaac 180 ttcttttcct ttgccataat aacctccatt caaatttgca gcttccgttccaggcacttc 240 tgaaccggga gaattttcag tcattcaaat ctctgtaaat cctgccgggggttttacggt 300 ccgctgctgt tttatgatag ttccgcggtt tccttcccat tgccgcacgcacttcgc 357 63 376 DNA Unknown Unknown Bacterium 63 tatacgtccaaagacagtat tgacattctg gagaagttct ttccggacga taaggaagcc 60 agggaagaaaaataagtagc tataagatat ttttaggtga tcaggccgga ttggaggaaa 120 tatatgataacattgaccga gaatgaaaag aggatggtat ttcagctgga aggctgcaac 180 agatatgatgcgatacagga gattgctgtg ttctgccagt atacccgtga ccatgacaaa 240 aggaacattgccgaaaatct gttaaagaaa cttcgtgaac tctcagaaaa tgactgtagg 300 gagctgatttgtgatattca gaaaaattat aatctgcctc ggaaagcaaa gactgttgga 360 gagatgattgctgtgc 376 64 626 DNA Unknown Unknown Bacterium 64 tgtagatctc ctccgccgcgcacagctcca ccagccggcc caggtacatc acccccacca 60 cgtcggagac atagcgcaccatggacaggt cgtgggcgat gaacagatag gccagccccc 120 gctcctgctg gtagtcccggaggaggttga ccacctgggc ctggatggac acatccagag 180 cggagatggg ctcgtcgcagatgaccagat cgggctggag gatcagcgcc cgggcgatgc 240 ccacccgctg gcgctggccgccggagaact cgtgggcgta gcgcccggcg tgctccgccg 300 tcagccccac ccgctccagcatggggtaga cgtagtcgtt ggcctcctgc ttggtcttga 360 ccacatggtg ggccagcaggggctcggcga tgatgtcccg gacggtcatc cgggcattga 420 gggaggcgta ggggtcctggaagatcatct gcatcttgcg gcgccggggc tggagctcct 480 tttgggacat gcgggtgatgtcctcgccgt ccagcacgat cttcccggcg gtggggtcgt 540 acatgcggat gatagaccgggcgcagctgg acttgccgca gcccgactcg cccaccaggc 600 ccagggtctc cccccggcggatggag 626 65 457 DNA Unknown Unknown Bacterium 65 tcagactttc agaatctcctgttttgccat attcccggtt atcgtcagtc aacgatgccg 60 ccgcatcgcc tctttcccccttgcaggctg aaaaatattc gctctgacag cctgcttact 120 ggccggattg agcgccagaggaaatcagga cacaaaaaag cagtgaaaaa attttcacag 180 aaaatttgca cgtactttttcaaattttcc acatccgttt tttcactgct ttttgatacc 240 ttaatcctgc atgagaacaatagtatattg ataaatttaa ggctggtact gctgttccag 300 aatctgttcc acattttccgccgtaacaat gcgatagggg agactcacat agatgtcatc 360 caccagcgct atatcctccggcagctctgt gccctgcccc agagaatacg caatttccag 420 aatactgtgc gcctgtccgggagcgtcgtt cagcact 457 66 364 DNA Unknown Unknown Bacterium 66cggcataggc gagttttgtt tggaattaca ttcgggcaag tctgcggaca agtcggaaat 60tataagaaat atagaaacca cgctttcgct caccgccgaa aacgacgggg aagagtttat 120aaatgctggc gcgagaatac gcgaaacgcg cgaggcgctg cacgcgcccc ttgcggcttt 180gcataaaaag cgtaggctgg gcgtttccgt ttacgagggg atagtttact atttgcaaaa 240taaatccgcg cccgagcttt taaatataga aactactttc tacgattctt taaccaaaca 300aaagttagag gattacgaaa atatgcttat taccgctcaa gcggcggcaa aggagtgcgg 360cgcc 364 67 616 DNA Unknown Unknown Bacterium 67 ggttcctgct gcttgcagcattcctcttcg cgagatcctc ccctcgcacg cacgcatggc 60 tgtgctccac gaaggtgtaccggcgctacg tcgccgcgtt caagcaagcc ggaggcattc 120 cgctttccac gaagatccggatagtcggcg tctcgtacgc catgatgggc gtcagcgcgc 180 tggtcgtcca gaagccgctggtgtgggcgg tcctggggtg cgtggccgtc ttcctcctgt 240 atctgatggc cgtccgcattccgaccatcg agcagaagcg tgtcgatcga gcccgggcag 300 aagacgtggc ctgagtcacggcaagccacc aagagcgccg gcgccgcagc catggcgcac 360 atggctcacc agaaggtcgaagccatggag gcgacgagac cccccgccta gtgcggcggg 420 agcagctatc agaaccgtgtcggagctggg cggtagaccg catcttcgac gagccgctgg 480 ccacgatgct gcctgcctctcatcatggcc aagcttcgct tcaagcatgc tgagcgcctg 540 ttttctcgca ccggacatttcgaaacgcgc tggatgcaag gatgcaacca aagacgacac 600 gaacgtgaaa catacg 616 68369 DNA Unknown Unknown Bacterium 68 gctgtataaa aacggcgtgc ttgcggaaaagaagcccgta agcttttaca gcgggctgaa 60 cgtggttacg ctgccgcttt acaccgatgaggagggcacc ttcgattacg agttgaaggt 120 tgaggcggcg gagccggacg gagatttttcgccgcataac aacgtttgct attttacgca 180 gaaggtgacc aacgagcgca aggtgctgtttttgggcggc tctgccgcgg atttggcggc 240 gggcaaaaga atttacggcg agaaggacgtaagctatata agcgacgtga agcaggtgcc 300 cgtgacggtg gaggatatgt gcggctatgacgaaattgtt ttaagcaact tcgacgtgcg 360 cacggtaag 369 69 604 DNA UnknownUnknown Bacterium 69 ggcctatgga gccgatgggg tctattggga aagggaaagcaaggacggtt ccgtgaaaaa 60 ggttggggaa cgctgccaga cctgtgcgga acggaaatatcaggacggtt ctgatgaaaa 120 tgtttccttt aaggcggcag cccatatttc cccggaagcagcaggcagcg cagtgcgcgc 180 ccatgaaggg gagcatgtgt ccaatgccta tacaaaggcggcaaagaatg atgggaaggt 240 tgtgtcagca tccgtcagca tccatacttc tgtctgcccggaatgtggca ggacgtatgt 300 atcgggggga accacatcca cccggattaa atacccggctgacccttatg aaaagagcaa 360 aaaggtgctt ggggaggaag aagccaaagg gaaaaatattgattatgcag cttaatttaa 420 ggaggtaaga atgatgaaga aattcagaac gaaaataacggcaatgatgt gcatgatgtt 480 ctgccttgtg gcggcagggg tgctcttaac ccctaatacggcatttgcga agaaaattac 540 ccgggacagc caggccgaat ccatggcaag gaaaaaagtaaaaggcggga ctgtcgtaga 600 aatc 604 70 282 DNA Unknown Unknown Bacterium70 ggccacaata caaccatcac ccagactaat tcccgtagaa ctatttacac ccaaaaggca 60atttttgccg atactaattg gctcactatt tccgccactt aaaacaccaa gtatgctagc 120cccaccgccg acatcgctcc cctcacctac gacaacacta gagctaatgc gcccttcatt 180catacaagca cccattgcac cagcattgaa attcacataa cttgctccgg gcatttgtgt 240ataaccacct ttgccaagat acgctccaaa acgggttttt cc 282 71 544 DNA UnknownUnknown Bacterium 71 ggatttattc ctttttatac ttttgtggct tccaaattttgaaatatacg tattttgaaa 60 ttattaagaa aatataaata catttttttg aaatttttatttaaaaatat attgacattt 120 agaaataaat attttataat acagttacct taatcaatctactttatcag cttcgcattt 180 tactgtctgg aggtaagctt gagcaacggt acaattgcgatttatgattc agaagcagac 240 tatgctttaa aacttgcaga atattttcgc ctgaaaaatggattaaacta ttctgtctcg 300 gtattcaccg attataattc ccttaagaat tatttatctgaaaatgatat tgatatactt 360 ttaatttctg aagaattttg tatgtatata gaagaattacagaatgtatc caatctgttt 420 atccttacag atggaaatat agatgctgcg ctaaagcagtatgcatccct atacaaatat 480 cagccagccg ataggatgct tcgggatatc atgtcctgttatgcactctc ttcttccaga 540 gaaa 544 72 664 DNA Unknown Unknown Bacterium72 gatactatat cttgtatact caaagcttat tgcgacagcc cctaggctga cctgttacat 60acaagcaaca ggtcatacgc cacttgtgca gcatacttcc tctgcacggg tctatgcata 120aaatcaaaat cgagtaggag atatttcttc ccctactcgc cgccggattc tccatttact 180taaatcgtac agctgttccg tatgccataa cttcggctgc gccttgcatt accgcagccg 240aagcataacg cacatttaaa accgcatctg caccaagctg ctctgcttcc tcaaccatcc 300gctttgttgc aagcgccctc gcttcattca tcatatccgt atacgctttt aactcgccgc 360caacaagcgt tttaaaactt tgtgtaatat ctttaccaaa gtttttactt tgaattgtac 420tgccctttac caaaccaagc atttctgttt cttttcctga tatataatca gtatttacta 480agatcatctt cttccccact ccttatttca ttcattcttt ccacaagcac atatacaaca 540acgccgatta gcgcaagtgg tatcactcca aacaaaattt tcgttatcaa caacactgac 600atactaaaac atatcgccgc ataaaaacaa aaataaatca ctggaataat agaaatcaca 660attg 664 73 613 DNA Unknown Unknown Bacterium 73 ggccaggccc gttatagggaatatctccat gaatctcata atccggataa tgaaccgcat 60 attcttttaa aatttcaatggccttgtccg cataggcctg ctctcctgta gccagccaaa 120 tcactgccat agagaatgccccttcataat tccccccatt gatcagtccc caccaggcac 180 tgtcataggg ttctcctgtaaatacctttt tacaggaagg gcatctgtgt ttatggggac 240 tgtttctgtc aaaatcaagctttaccgagc agtccggaca atagtaataa agcgtccagt 300 tggcgatccc cctttcgggaaccattatct caccattaaa gatctcatct acttcctttt 360 ttagccgctt aattgtggcaggataacggc tgcttctctc tctcagatgt tttctctctt 420 catccgtaaa ctgaatcattttccttctcc ccttttatca caatttttcc tgtttctgat 480 acagccaaag agcccctttcctttaaacgc tgtccggctt caaagtttct ttaaactctg 540 taggcgtaca cccgtagaactttttaaagc tgctggaaaa ataccgctga ttgtcatacc 600 cgcagcgttc tgc 613 74514 DNA Unknown Unknown Bacterium 74 ttcatcactt aatgaataaa gcactggcaggtccattgga aggaaacggc gtatctctgt 60 tttacagtca aattggtgta atgccttgttggcaacactt gaaagatatt ccatacgctg 120 gtattcatat ggctgtaatt cttctaatacaaagtccatg ctttcttcct ggaatggcgc 180 aagcggaagt gaatacatat gtgcaagtttccttatcagt tcttcatcat atacataccc 240 tgcacatatt aaaagccttc cctgtgccatatataaaggc cttaactggt taaatcttga 300 gactgagcca atccagtctg cttcacccactttctttaaa agctctcctg tgtaagtgcc 360 ctcgcttgtt tcaaatggca gataatctataaacaagtgg aaaagcctgt catcccatac 420 tgacattgat ttaataagct tgtttttatctgtaataacc tctctgtctg caagcatttc 480 ccccctgtca gtcattctgc aataatccaaatca 514 75 1377 DNA Unknown Unknown Bacterium 75 gtagtacagc acaatcttagagcaatgaat tctaacagaa tgttaggcat cacacaggga 60 tctttaaaca aatctacagagaagctctca tcaggctaca aggtaaacag ggcagcagat 120 gatgcagcgg gtctttcaatttccgagaaa atgagaaaac agatcagagg actgtcacag 180 gcatctttga atgctgaggatggtatcagt gcagtgcaga ccgcagaggg cgcattgaca 240 gaagttcatg acatgttgcagagaatgaac gagctggcag taaaggctgc aaacggcaca 300 aactctacat cagaccgtcagacaattcag gacgaggtag accagctcct cacagaaatc 360 gaccgtgtcg cagagaccaccaaattcaat gagctgtata cattgaaggg tgatgaggac 420 aaggtgacaa gatatctttcagcacatgac gcaggtatag aaggaacctt gacacagggc 480 gctacaaacg cgacattttcaatggaccag ttaaagtttg gcgacaccat catgatcgca 540 ggcagagagt accatatcagcggaaccaag gcagagcagg cagcaatcat tacggcttct 600 gtgaagattg gacagcaggttacgattgat ggaatcatgt atacctgttc atcagtaagc 660 aatgctgaca aatttgaactgaaaagtgag gatttgattg caaaactcga cacttcaagc 720 ctgagtatta tgtcagtgaatggcaagacc tactacggcg caggcatcac agatgacagg 780 actgttgtaa gttcaattggtgcatacaag ctgattcaga aggagctcgg actggcaagc 840 agcattggtg cagacggcgcaacacaggct tcggtaaatg ccggagtaga tggcaagact 900 ttgatgaagc cgagttttgagggcaaatgg gtatttagta tcgacaaggg aagcgttcag 960 gtacgcgagg acattgatttcagcctccat gtaggtgcag atgccgacat gaacaacaag 1020 attgcggtga agatcggagcgcttgacacg aagggacttg gtatccaagg actgaatgta 1080 aaggatacga caggcgcagcagcgacctac gcgattgatt cgattgcgga cgcagtggca 1140 agaatttctg cgcagcgctctttactcggt gcagtgcaga accggttaga gcacacgatc 1200 aacaacttgg ataacgttgtagagaacaca accgccgcag agagccagat ccgtgataca 1260 gacatggcga cagagatggtgaagtactct aataacaacg tacttgcaca ggcaggccag 1320 tcaatgttag cacagtctaatcaggcaaat cagggtgtac ttcagctctt acagtaa 1377 76 450 DNA Unknown UnknownBacterium 76 gtagtacagc acaatcttag agcaatgaat tctaacagaa tgttaggcatcacacaggga 60 tctttaaaca aatcgacaga gaagctatca tcaggctaca aggtaaacagggcagcagat 120 gatgcagcgg gtctttcaat ttccgagaaa atgagaaaac agatcagaggactgtcacag 180 gcatctttga atgctgagga tggtatcagt gcagtgcaga ccgcagagggcgcattgaca 240 gaagttcatg acatgttgca gagaatgaac gagctggcag taaaggctgcaaacggcaca 300 aactctacat cagaccgtca gacaattcag gacgaggtag accagctcctcacagaaatc 360 gaccgtgtcg cagagaccac caaattcaat gagctgtata cattgaagggtgatgaggac 420 aaggtgacaa gatatctttc agcacattaa 450 77 990 DNA UnknownUnknown Bacterium 77 gtagtacagc acaatcttag agcaatgaat tctaacagaatgttaggcat cacacaggga 60 tctttaaaca aatctacaga gaagctctca tcaggctacaaggtaaacag ggcagcagat 120 gatgcagcgg gtctttcaat ttccgagaaa atgagaaaacagatcagagg actgtcacag 180 gcatctttga atgctgagga tggtatcagt gcagtgcagaccgcagaggg cgcattgaca 240 gaagttcatg acatgttgca gagaatgaac gagctggcagtaaaggctgc aaacggcaca 300 aactctacat cagaccgtca gacaattcag gacgaggtagaccagctcct cacagaaatc 360 gaccgtgtcg cagagaccac caaattcaat gagctgtatacattgaaggg tgatgaggac 420 aaggtgacaa gatatctttc agcacatgac gcaggtatagaaggaacctt gacacagggc 480 gctacaaacg cgacattttc aatggaccag ttaaagtttggcgacaccat catgatcgca 540 ggcagagagt accatatcag cggaaccaag gcagagcaggcagcaatcat tacggcttct 600 gtgaagattg gacagcaggt tacgattgat ggaatcatgtatacctgttc atcagtaagc 660 aatgctgaca aatttgaact gaaaagtgag gatttgattgcaaaactcga cacttcaagc 720 ctgagtatta tgtcagtgaa tggcaagacc tactacggcgcaggcatcac agatgacagg 780 actgttgtaa gttcaattgg tgcatacaag ctgattcagaaggagctcgg actggcaagc 840 agcattggtg cagacggcgc aacacaggct tcggtaaatgccggagtaga tggcaagact 900 ttgatgaagc cgagttttga gggcaaatgg gtatttagtatcgacaaggg aagcgttcag 960 gtacgcgagg acattgattt cagcctctaa 990 78 399DNA Unknown Unknown Bacterium 78 ttcagcctcc atgtaggtgc agatgccgacatgaacaaca agattgcggt gaagatcgga 60 gcgcttgaca cgaagggact tggtatccaaggactgaatg taaaggatac gacaggcgca 120 gcagcgacct acgcgattga ttcgattgcggacgcagtgg caagaatttc tgcgcagcgc 180 tctttactcg gtgcagtgca gaaccggttagagcacacga tcaacaactt ggataacgtt 240 gtagagaaca caaccgccgc agagagccagatccgtgata cagacatggc gacagagatg 300 gtgaagtact ctaataacaa cgtacttgcacaggcaggcc agtcaatgtt agcacagtct 360 aatcaggcaa atcagggtgt acttcagctcttacagtaa 399 79 458 PRT Unknown Unknown Bacterium 79 Val Val Gln HisAsn Leu Arg Ala Met Asn Ser Asn Arg Met Leu Gly 5 10 15 Ile Thr Gln GlySer Leu Asn Lys Ser Thr Glu Lys Leu Ser Ser Gly 20 25 30 Tyr Lys Val AsnArg Ala Ala Asp Asp Ala Ala Gly Leu Ser Ile Ser 35 40 45 Glu Lys Met ArgLys Gln Ile Arg Gly Leu Ser Gln Ala Ser Leu Asn 50 55 60 Ala Glu Asp GlyIle Ser Ala Val Gln Thr Ala Glu Gly Ala Leu Thr 65 70 75 80 Glu Val HisAsp Met Leu Gln Arg Met Asn Glu Leu Ala Val Lys Ala 85 90 95 Ala Asn GlyThr Asn Ser Thr Ser Asp Arg Gln Thr Ile Gln Asp Glu 100 105 110 Val AspGln Leu Leu Thr Glu Ile Asp Arg Val Ala Glu Thr Thr Lys 115 120 125 PheAsn Glu Leu Tyr Thr Leu Lys Gly Asp Glu Asp Lys Val Thr Arg 130 135 140Tyr Leu Ser Ala His Asp Ala Gly Ile Glu Gly Thr Leu Thr Gln Gly 145 150155 160 Ala Thr Asn Ala Thr Phe Ser Met Asp Gln Leu Lys Phe Gly Asp Thr165 170 175 Ile Met Ile Ala Gly Arg Glu Tyr His Ile Ser Gly Thr Lys AlaGlu 180 185 190 Gln Ala Ala Ile Ile Thr Ala Ser Val Lys Ile Gly Gln GlnVal Thr 195 200 205 Ile Asp Gly Ile Met Tyr Thr Cys Ser Ser Val Ser AsnAla Asp Lys 210 215 220 Phe Glu Leu Lys Ser Glu Asp Leu Ile Ala Lys LeuAsp Thr Ser Ser 225 230 235 240 Leu Ser Ile Met Ser Val Asn Gly Lys ThrTyr Tyr Gly Ala Gly Ile 245 250 255 Thr Asp Asp Arg Thr Val Val Ser SerIle Gly Ala Tyr Lys Leu Ile 260 265 270 Gln Lys Glu Leu Gly Leu Ala SerSer Ile Gly Ala Asp Gly Ala Thr 275 280 285 Gln Ala Ser Val Asn Ala GlyVal Asp Gly Lys Thr Leu Met Lys Pro 290 295 300 Ser Phe Glu Gly Lys TrpVal Phe Ser Ile Asp Lys Gly Ser Val Gln 305 310 315 320 Val Arg Glu AspIle Asp Phe Ser Leu His Val Gly Ala Asp Ala Asp 325 330 335 Met Asn AsnLys Ile Ala Val Lys Ile Gly Ala Leu Asp Thr Lys Gly 340 345 350 Leu GlyIle Gln Gly Leu Asn Val Lys Asp Thr Thr Gly Ala Ala Ala 355 360 365 ThrTyr Ala Ile Asp Ser Ile Ala Asp Ala Val Ala Arg Ile Ser Ala 370 375 380Gln Arg Ser Leu Leu Gly Ala Val Gln Asn Arg Leu Glu His Thr Ile 385 390395 400 Asn Asn Leu Asp Asn Val Val Glu Asn Thr Thr Ala Ala Glu Ser Gln405 410 415 Ile Arg Asp Thr Asp Met Ala Thr Glu Met Val Lys Tyr Ser AsnAsn 420 425 430 Asn Val Leu Ala Gln Ala Gly Gln Ser Met Leu Ala Gln SerAsn Gln 435 440 445 Ala Asn Gln Gly Val Leu Gln Leu Leu Gln 450 455 80149 PRT Unknown Unknown Bacterium 80 Val Val Gln His Asn Leu Arg Ala MetAsn Ser Asn Arg Met Leu Gly 5 10 15 Ile Thr Gln Gly Ser Leu Asn Lys SerThr Glu Lys Leu Ser Ser Gly 20 25 30 Tyr Lys Val Asn Arg Ala Ala Asp AspAla Ala Gly Leu Ser Ile Ser 35 40 45 Glu Lys Met Arg Lys Gln Ile Arg GlyLeu Ser Gln Ala Ser Leu Asn 50 55 60 Ala Glu Asp Gly Ile Ser Ala Val GlnThr Ala Glu Gly Ala Leu Thr 65 70 75 80 Glu Val His Asp Met Leu Gln ArgMet Asn Glu Leu Ala Val Lys Ala 85 90 95 Ala Asn Gly Thr Asn Ser Thr SerAsp Arg Gln Thr Ile Gln Asp Glu 100 105 110 Val Asp Gln Leu Leu Thr GluIle Asp Arg Val Ala Glu Thr Thr Lys 115 120 125 Phe Asn Glu Leu Tyr ThrLeu Lys Gly Asp Glu Asp Lys Val Thr Arg 130 135 140 Tyr Leu Ser Ala His145 81 329 PRT Unknown Unknown Bacterium 81 Val Val Gln His Asn Leu ArgAla Met Asn Ser Asn Arg Met Leu Gly 5 10 15 Ile Thr Gln Gly Ser Leu AsnLys Ser Thr Glu Lys Leu Ser Ser Gly 20 25 30 Tyr Lys Val Asn Arg Ala AlaAsp Asp Ala Ala Gly Leu Ser Ile Ser 35 40 45 Glu Lys Met Arg Lys Gln IleArg Gly Leu Ser Gln Ala Ser Leu Asn 50 55 60 Ala Glu Asp Gly Ile Ser AlaVal Gln Thr Ala Glu Gly Ala Leu Thr 65 70 75 80 Glu Val His Asp Met LeuGln Arg Met Asn Glu Leu Ala Val Lys Ala 85 90 95 Ala Asn Gly Thr Asn SerThr Ser Asp Arg Gln Thr Ile Gln Asp Glu 100 105 110 Val Asp Gln Leu LeuThr Glu Ile Asp Arg Val Ala Glu Thr Thr Lys 115 120 125 Phe Asn Glu LeuTyr Thr Leu Lys Gly Asp Glu Asp Lys Val Thr Arg 130 135 140 Tyr Leu SerAla His Asp Ala Gly Ile Glu Gly Thr Leu Thr Gln Gly 145 150 155 160 AlaThr Asn Ala Thr Phe Ser Met Asp Gln Leu Lys Phe Gly Asp Thr 165 170 175Ile Met Ile Ala Gly Arg Glu Tyr His Ile Ser Gly Thr Lys Ala Glu 180 185190 Gln Ala Ala Ile Ile Thr Ala Ser Val Lys Ile Gly Gln Gln Val Thr 195200 205 Ile Asp Gly Ile Met Tyr Thr Cys Ser Ser Val Ser Asn Ala Asp Lys210 215 220 Phe Glu Leu Lys Ser Glu Asp Leu Ile Ala Lys Leu Asp Thr SerSer 225 230 235 240 Leu Ser Ile Met Ser Val Asn Gly Lys Thr Tyr Tyr GlyAla Gly Ile 245 250 255 Thr Asp Asp Arg Thr Val Val Ser Ser Ile Gly AlaTyr Lys Leu Ile 260 265 270 Gln Lys Glu Leu Gly Leu Ala Ser Ser Ile GlyAla Asp Gly Ala Thr 275 280 285 Gln Ala Ser Val Asn Ala Gly Val Asp GlyLys Thr Leu Met Lys Pro 290 295 300 Ser Phe Glu Gly Lys Trp Val Phe SerIle Asp Lys Gly Ser Val Gln 305 310 315 320 Val Arg Glu Asp Ile Asp PheSer Leu 325 82 129 PRT Unknown Unknown Bacterium 82 Phe Ser Leu His ValGly Ala Asp Ala Asp Met Asn Asn Lys Ile Ala 5 10 15 Val Lys Ile Gly AlaLeu Asp Thr Lys Gly Leu Gly Ile Gln Gly Leu 20 25 30 Asn Val Lys Asp ThrThr Gly Ala Ala Ala Thr Tyr Ala Ile Asp Ser 35 40 45 Ile Ala Asp Ala ValAla Arg Ile Ser Ala Gln Arg Ser Leu Leu Gly 50 55 60 Ala Val Gln Asn ArgLeu Glu His Thr Ile Asn Asn Leu Asp Asn Val 65 70 75 80 Val Glu Asn ThrThr Ala Ala Glu Ser Gln Ile Arg Asp Thr Asp Met 85 90 95 Ala Thr Glu MetVal Lys Tyr Ser Asn Asn Asn Val Leu Ala Gln Ala 100 105 110 Gly Gln SerMet Leu Ala Gln Ser Asn Gln Ala Asn Gln Gly Val Leu 115 120 125 Gln 831542 DNA Helicobacter bilis 83 atgagtttta ggataaatac aaatatcgcggcactcaatg cgcataccat cggtgtgcaa 60 aacaataggg caatagccaa ctcgttagagaagttaagct ctggtttgag gattaacaag 120 gcagcagatg atgcttcagg tatgtcaatcgcagatagtt tgcgtagcca agcaagttca 180 ttaggacagg caatcggcaa cgcaaatgatgcgattggta tgattcagat tgcggataaa 240 gcaatggatg agcagctaaa gattcttgataccgtaaagg ttaaagcaat ccaagcagct 300 caagatgggc aaactactga atcaagacgcgcgttgcaaa acgatattgt gcgactctta 360 gaagagcttg ataatatcgc caatacaacaagctataacg ggcagcaaat gctatctggt 420 gctttctcta acaaagagtt ccaaatcggtgcgtattcta atacaacagt gaaagcttca 480 atcggtccaa caagctcaga taaaatcggacatgtaagac ttgaaagctc atctgtaaca 540 ggtattggta tgcttgctag tgctggtgctaagaatctta aagaggtagc attgaaattc 600 cgccaagttg atggtaagaa agactacaagcttgagactg cagtcatttc tacaagtgct 660 ggcacaggta ttggtgtatt agcagatactatcaacaaat tctctgatac actcggtgtg 720 cgtgcgtatg caacggtgct tgggactggtggtgtgccgg tgcaatctgg aacagtgcat 780 ggcttagttg taaatggcac aactattgggacaatcaatg atgtgcgtaa aaatgatgct 840 gatggtagat tgattaatgc ctttaactcaattaaagaaa gaacaggtgt agaagcgtat 900 gtggatatcg aaggtagatt aaaccttagaagtcttgatg gtcgtgctat atctgtgcat 960 gctgaaggta aaacaggtgc ggtgcttggtggcggtagct ttgctggagt atctgggaca 1020 aatcacgcta tcgtgggtcg tataagccttgttaggacag atgcaagaga tattattgta 1080 tctgggacaa actttagtag tgttggtttccactctgctc aaggtatcgc acaatacact 1140 gtgaatttgc gttctgttcg cggtaatatggacgcaaata tcgcaagtgc aagcggtgca 1200 aacgcaaatg cggctcaagc ggtgcagaataaagatggta tcggcgcagg tgttacttcg 1260 cttcgcggtg cgatggtcgt tatggatatggcagaatctg ctacaagaca gcttgataaa 1320 atccgtgctg acatgggttc tgtgcaaatgcagcttgttg ctacaatcaa caacatttct 1380 atcacgcaag ttaatgttaa agcggctgaaagtcaaatta gagatgtgga tttcgcacaa 1440 gaatctgcga cattctctaa gcataacatcttggctcaat ctggtagctt tgctatggct 1500 caagctaacg cagtgcaaca aaatgtcttaagacttttgc aa 1542 84 514 PRT Helicobacter bilis 84 Met Ser Phe Arg IleAsn Thr Asn Ile Ala Ala Leu Asn Ala His Thr 5 10 15 Ile Gly Val Gln AsnAsn Arg Ala Ile Ala Asn Ser Leu Glu Lys Leu 20 25 30 Ser Ser Gly Leu ArgIle Asn Lys Ala Ala Asp Asp Ala Ser Gly Met 35 40 45 Ser Ile Ala Asp SerLeu Arg Ser Gln Ala Ser Ser Leu Gly Gln Ala 50 55 60 Ile Gly Asn Ala AsnAsp Ala Ile Gly Met Ile Gln Ile Ala Asp Lys 65 70 75 80 Ala Met Asp GluGln Leu Lys Ile Leu Asp Thr Val Lys Val Lys Ala 85 90 95 Ile Gln Ala AlaGln Asp Gly Gln Thr Thr Glu Ser Arg Arg Ala Leu 100 105 110 Gln Asn AspIle Val Arg Leu Leu Glu Glu Leu Asp Asn Ile Ala Asn 115 120 125 Thr ThrSer Tyr Asn Gly Gln Gln Met Leu Ser Gly Ala Phe Ser Asn 130 135 140 LysGlu Phe Gln Ile Gly Ala Tyr Ser Asn Thr Thr Val Lys Ala Ser 145 150 155160 Ile Gly Pro Thr Ser Ser Asp Lys Ile Gly His Val Arg Leu Glu Ser 165170 175 Ser Ser Val Thr Gly Ile Gly Met Leu Ala Ser Ala Gly Ala Lys Asn180 185 190 Leu Lys Glu Val Ala Leu Lys Phe Arg Gln Val Asp Gly Lys LysAsp 195 200 205 Tyr Lys Leu Glu Thr Ala Val Ile Ser Thr Ser Ala Gly ThrGly Ile 210 215 220 Gly Val Leu Ala Asp Thr Ile Asn Lys Phe Ser Asp ThrLeu Gly Val 225 230 235 240 Arg Ala Tyr Ala Thr Val Leu Gly Thr Gly GlyVal Pro Val Gln Ser 245 250 255 Gly Thr Val His Gly Leu Val Val Asn GlyThr Thr Ile Gly Thr Ile 260 265 270 Asn Asp Val Arg Lys Asn Asp Ala AspGly Arg Leu Ile Asn Ala Phe 275 280 285 Asn Ser Ile Lys Glu Arg Thr GlyVal Glu Ala Tyr Val Asp Ile Glu 290 295 300 Gly Arg Leu Asn Leu Arg SerLeu Asp Gly Arg Ala Ile Ser Val His 305 310 315 320 Ala Glu Gly Lys ThrGly Ala Val Leu Gly Gly Gly Ser Phe Ala Gly 325 330 335 Val Ser Gly ThrAsn His Ala Ile Val Gly Arg Ile Ser Leu Val Arg 340 345 350 Thr Asp AlaArg Asp Ile Ile Val Ser Gly Thr Asn Phe Ser Ser Val 355 360 365 Gly PheHis Ser Ala Gln Gly Ile Ala Gln Tyr Thr Val Asn Leu Arg 370 375 380 SerVal Arg Gly Asn Met Asp Ala Asn Ile Ala Ser Ala Ser Gly Ala 385 390 395400 Asn Ala Asn Ala Ala Gln Ala Val Gln Asn Lys Asp Gly Ile Gly Ala 405410 415 Gly Val Thr Ser Leu Arg Gly Ala Met Val Val Met Asp Met Ala Glu420 425 430 Ser Ala Thr Arg Gln Leu Asp Lys Ile Arg Ala Asp Met Gly SerVal 435 440 445 Gln Met Gln Leu Val Ala Thr Ile Asn Asn Ile Ser Ile ThrGln Val 450 455 460 Asn Val Lys Ala Ala Glu Ser Gln Ile Arg Asp Val AspPhe Ala Gln 465 470 475 480 Glu Ser Ala Thr Phe Ser Lys His Asn Ile LeuAla Gln Ser Gly Ser 485 490 495 Phe Ala Met Ala Gln Ala Asn Ala Val GlnGln Asn Val Leu Arg Leu 500 505 510 Leu Gln 85 1494 DNA Unknown UnknownBacterium 85 ggaggtatta ttatggtagt acagcacaat ttacaggcaa tgaactctaacagaatgtta 60 ggcatcacac agaagacagc atctaagtct acagaaaagt tatcttcaggttacgcaatc 120 aaccgcgcag cagacaacgc agcaggtctt gctatttctg agaagatgagaaagcagatc 180 agaggactta cacaggcttc tacaaatgct gaggacggca tcagctctgtacagacagca 240 gaaggcgctt tgacagaagt gcatgatatg cttcagagaa tgaacgagctggcaattcag 300 gcagcaaacg gcacaaactc agaagatgac cgctcataca ttcaggacgaaattgaccag 360 ctgacacagg aaatcgatcg tgttgctgag acaacaaagt tcaatgagacatatctcttg 420 aagggtgaca caaagaacgt tgacgctatg gactatacat atagctataaggcagttaca 480 acgaatactg tagcaagagc ttcggtttta gcagcagaga acacagctacaggtatgtca 540 gttagtattt catttgctgc aaacagcggc aaggttactg cagctgactctaacaacctt 600 gcaaaggcta tcagagatca gggcttcaca atcacaacat ctacccagaatggtaaggtt 660 gtttacggtc ttgagctgaa cggaagcgat gcaaaggcaa actatacagtttcaacagta 720 agtatggaag ctggtacatt caagatcctg aattctaata agcaggttgttgcatctgta 780 acaatatcta caacagctag ctttaaaaag gtatctggta tgtcacagatcgttacggcg 840 tactctgtat cagcagctta tgcgacgggt gatgtatact ctctctatgacgcagacgga 900 aatgcaattt cagcaaacaa gctggataag tactttacgg caggcggcgctacagaggca 960 ggcggaatag ctactacact ttcagcaaac tctggtgtgc ctaaggtttatgacgtactc 1020 ggaaaagagg tttctgcagt aagcattgca agtactttag taacagcagttaaggataag 1080 acggctgcat tgaagatgaa cttccatgta ggtgctgacg gaacagataacaacaagatt 1140 aagatcaaca ttgaggctat gacagctaag agtcttggag ttaacggtctgaaggtgagc 1200 ggttcgagcg gaacaaacgc tacaaacgct atcgagataa tcgctggcgctatcaagaag 1260 gtttctacac agagatctgc tcttggtgcg gttcagaaca gattagagcacacaatcaac 1320 aacttggata acatcgttga gaacacaaca gcagctgagt caggaatccgcgatacagat 1380 atggctacag agatggttaa gtactctaac gctaatatcc tttcacaggcaggtcagtct 1440 atgcttgcac agtctaacca gtctaaccag ggtgtacttc agctcttacagtaa 1494 86 493 PRT Unknown Unknown Bacterium 86 Met Val Val Gln HisAsn Leu Gln Ala Met Asn Ser Asn Arg Met Leu 5 10 15 Gly Ile Thr Gln LysThr Ala Ser Lys Ser Thr Glu Lys Leu Ser Ser 20 25 30 Gly Tyr Ala Ile AsnArg Ala Ala Asp Asn Ala Ala Gly Leu Ala Ile 35 40 45 Ser Glu Lys Met ArgLys Gln Ile Arg Gly Leu Thr Gln Ala Ser Thr 50 55 60 Asn Ala Glu Asp GlyIle Ser Ser Val Gln Thr Ala Glu Gly Ala Leu 65 70 75 80 Thr Glu Val HisAsp Met Leu Gln Arg Met Asn Glu Leu Ala Ile Gln 85 90 95 Ala Ala Asn GlyThr Asn Ser Glu Asp Asp Arg Ser Tyr Ile Gln Asp 100 105 110 Glu Ile AspGln Leu Thr Gln Glu Ile Asp Arg Val Ala Glu Thr Thr 115 120 125 Lys PheAsn Glu Thr Tyr Leu Leu Lys Gly Asp Thr Lys Asn Val Asp 130 135 140 AlaMet Asp Tyr Thr Tyr Ser Tyr Lys Ala Val Thr Thr Asn Thr Val 145 150 155160 Ala Arg Ala Ser Val Leu Ala Ala Glu Asn Thr Ala Thr Gly Met Ser 165170 175 Val Ser Ile Ser Phe Ala Ala Asn Ser Gly Lys Val Thr Ala Ala Asp180 185 190 Ser Asn Asn Leu Ala Lys Ala Ile Arg Asp Gln Gly Phe Thr IleThr 195 200 205 Thr Ser Thr Gln Asn Gly Lys Val Val Tyr Gly Leu Glu LeuAsn Gly 210 215 220 Ser Asp Ala Lys Ala Asn Tyr Thr Val Ser Thr Val SerMet Glu Ala 225 230 235 240 Gly Thr Phe Lys Ile Leu Asn Ser Asn Lys GlnVal Val Ala Ser Val 245 250 255 Thr Ile Ser Thr Thr Ala Ser Phe Lys LysVal Ser Gly Met Ser Gln 260 265 270 Ile Val Thr Ala Tyr Ser Val Ser AlaAla Tyr Ala Thr Gly Asp Val 275 280 285 Tyr Ser Leu Tyr Asp Ala Asp GlyAsn Ala Ile Ser Ala Asn Lys Leu 290 295 300 Asp Lys Tyr Phe Thr Ala GlyGly Ala Thr Glu Ala Gly Gly Ile Ala 305 310 315 320 Thr Thr Leu Ser AlaAsn Ser Gly Val Pro Lys Val Tyr Asp Val Leu 325 330 335 Gly Lys Glu ValSer Ala Val Ser Ile Ala Ser Thr Leu Val Thr Ala 340 345 350 Val Lys AspLys Thr Ala Ala Leu Lys Met Asn Phe His Val Gly Ala 355 360 365 Asp GlyThr Asp Asn Asn Lys Ile Lys Ile Asn Ile Glu Ala Met Thr 370 375 380 AlaLys Ser Leu Gly Val Asn Gly Leu Lys Val Ser Gly Ser Ser Gly 385 390 395400 Thr Asn Ala Thr Asn Ala Ile Glu Ile Ile Ala Gly Ala Ile Lys Lys 405410 415 Val Ser Thr Gln Arg Ser Ala Leu Gly Ala Val Gln Asn Arg Leu Glu420 425 430 His Thr Ile Asn Asn Leu Asp Asn Ile Val Glu Asn Thr Thr AlaAla 435 440 445 Glu Ser Gly Ile Arg Asp Thr Asp Met Ala Thr Glu Met ValLys Tyr 450 455 460 Ser Asn Ala Asn Ile Leu Ser Gln Ala Gly Gln Ser MetLeu Ala Gln 465 470 475 480 Ser Asn Gln Ser Asn Gln Gly Val Leu Gln LeuLeu Gln 485 490 87 2019 DNA Unknown Unknown Bacterium 87 gtgtgtggtccacggcgcgg ccttcaccgg cggtgagcgc acggaccagg tgctggcgga 60 cttcaccgccccggaggacg gtcttctcca catcctctgc ctccacggcg acgtcttcag 120 ccaggacagcgtctacggcc ccatcacccg gccccagatc gcccgcagcg gcgcggatta 180 cctggccctgggccacgtcc accagtgcag cggcatccag cgccaggggg acaccccctg 240 ggcctaccccggctgtcccg agggccgggg gttcgacgag ctgggggaca agggtgtgct 300 ggcggggacggtggatcggg gcggggcggc ggatctgcgc ttcgtgcccc tgtgccgccg 360 ccggtaccggattctggagg cggacgtgac ggaccgggac cccggcgagg ccctggaggc 420 cgtgatccccgccaccgccg ccatggacgt gtgccgcgtg ctcctcaccg gcgaaatcgg 480 ggagccgggcgcggatctgg cggacctgga gcgccggtac caggaccgct tctacgccct 540 ggagctccgggaccgcaccc gggccgccca gaacctgtgg gcccgggccg gagaagactt 600 ccatggaagagcggaagagg caaagaggaa gagcagcaag aataaccccg ttgagaagat 660 ggcgggtaagatgggtaagg aataggtaaa tatgcaggga tgcctaaggt tcctgcttat 720 tataaatagccaatccgcag ggcggggagg ctttgtggaa gccaatttgc acagcttggc 780 ttatcatggaaaagctgctg tgtgaagagg aaagagttaa aacatatatg tgcgcaaacg 840 gatttgcggcatggctgttg gcttgcaaag caagtggcag taacaggcct gctgaaggga 900 agccataaataggcaggcat atcaaaaaca aggaggaaaa atattatggc aatggtaatc 960 caacacaatcttacagcaat gaattctaac cgtcagttgg gagtaatcac tagcgggcag 1020 gcgaaatcttctgaaaagtt gtcatctgga tataggatta atcgtgcagc agatgatgca 1080 gcagggttaaagatttctga gaaaatgagg agccaggttc gtggattgaa tagggcatcc 1140 acaaatgcacaggatggtat ctctttgatt cagacggcag aaggtgcgct gaatgaagca 1200 cattccatccttcagcgtat gcacgagttg gcagtccaag gtgcaaatga tacaaaccag 1260 gatattgaccgtgaggcgat agacgaggaa ttggctgcat taacccaaga acttgatagg 1320 atttctgaaacaacacagtt taataaacag aatctattgg atggaagttt tcaggataag 1380 aatctccatgtaggtgcgaa tgcaaatcag aaaatcagca tcaagattga taatatggat 1440 gcagcggcgcttgggttaaa agattttgca tactataaag gcacagaaac agtatcttat 1500 tctaaaatgacatatatggg tgtaagttat acttatgata cgtcaaaaag tgatgcggca 1560 aatagaagtgcctttaaagc cctgttaaag tcagcaggga aagcagcatt tgtggatggg 1620 atgatagcacttcattcaga tggaaagtat tatttgagtg atacaactac aaattatacc 1680 accctttcagcagcacgtgc aaacggtaag tctaagcttg gtatacaata tgctggtttg 1740 gcaagtgcccaatggtcttc tctgctgaaa aatgcaagaa aatcttcaac aacgataggg 1800 gcatttggaagcaaagttac attctccagt cccactgttt cagattatga tagggcaaat 1860 gccacattgcaggcagttca ggcggctatt aatattgtat ctacacagcg ttctgcactt 1920 ggtgcaattcagaatcgttt agagcataca gtggcaaatc ttgataatgt agcagaaaat 1980 acacaggcagctgaatctag gattcgtgac acggatatg 2019 88 358 PRT Unknown UnknownBacterium 88 Met Ala Met Val Ile Gln His Asn Leu Thr Ala Met Asn Ser AsnArg 5 10 15 Gln Leu Gly Val Ile Thr Ser Gly Gln Ala Lys Ser Ser Glu LysLeu 20 25 30 Ser Ser Gly Tyr Arg Ile Asn Arg Ala Ala Asp Asp Ala Ala GlyLeu 35 40 45 Lys Ile Ser Glu Lys Met Arg Ser Gln Val Arg Gly Leu Asn ArgAla 50 55 60 Ser Thr Asn Ala Gln Asp Gly Ile Ser Leu Ile Gln Thr Ala GluGly 65 70 75 80 Ala Leu Asn Glu Ala His Ser Ile Leu Gln Arg Met His GluLeu Ala 85 90 95 Val Gln Gly Ala Asn Asp Thr Asn Gln Asp Ile Asp Arg GluAla Ile 100 105 110 Asp Glu Glu Leu Ala Ala Leu Thr Gln Glu Leu Asp ArgIle Ser Glu 115 120 125 Thr Thr Gln Phe Asn Lys Gln Asn Leu Leu Asp GlySer Phe Gln Asp 130 135 140 Lys Asn Leu His Val Gly Ala Asn Ala Asn GlnLys Ile Ser Ile Lys 145 150 155 160 Ile Asp Asn Met Asp Ala Ala Ala LeuGly Leu Lys Asp Phe Ala Tyr 165 170 175 Tyr Lys Gly Thr Glu Thr Val SerTyr Ser Lys Met Thr Tyr Met Gly 180 185 190 Val Ser Tyr Thr Tyr Asp ThrSer Lys Ser Asp Ala Ala Asn Arg Ser 195 200 205 Ala Phe Lys Ala Leu LeuLys Ser Ala Gly Lys Ala Ala Phe Val Asp 210 215 220 Gly Met Ile Ala LeuHis Ser Asp Gly Lys Tyr Tyr Leu Ser Asp Thr 225 230 235 240 Thr Thr AsnTyr Thr Thr Leu Ser Ala Ala Arg Ala Asn Gly Lys Ser 245 250 255 Lys LeuGly Ile Gln Tyr Ala Gly Leu Ala Ser Ala Gln Trp Ser Ser 260 265 270 LeuLeu Lys Asn Ala Arg Lys Ser Ser Thr Thr Ile Gly Ala Phe Gly 275 280 285Ser Lys Val Thr Phe Ser Ser Pro Thr Val Ser Asp Tyr Asp Arg Ala 290 295300 Asn Ala Thr Leu Gln Ala Val Gln Ala Ala Ile Asn Ile Val Ser Thr 305310 315 320 Gln Arg Ser Ala Leu Gly Ala Ile Gln Asn Arg Leu Glu His ThrVal 325 330 335 Ala Asn Leu Asp Asn Val Ala Glu Asn Thr Gln Ala Ala GluSer Arg 340 345 350 Ile Arg Asp Thr Asp Met 355 89 227 PRT UnknownUnknown Bacterium 89 Cys Val Val His Gly Ala Ala Phe Thr Gly Gly Glu ArgThr Asp Gln 5 10 15 Val Leu Ala Asp Phe Thr Ala Pro Glu Asp Gly Leu LeuHis Ile Leu 20 25 30 Cys Leu His Gly Asp Val Phe Ser Gln Asp Ser Val TyrGly Pro Ile 35 40 45 Thr Arg Pro Gln Ile Ala Arg Ser Gly Ala Asp Tyr LeuAla Leu Gly 50 55 60 His Val His Gln Cys Ser Gly Ile Gln Arg Gln Gly AspThr Pro Trp 65 70 75 80 Ala Tyr Pro Gly Cys Pro Glu Gly Arg Gly Phe AspGlu Leu Gly Asp 85 90 95 Lys Gly Val Leu Ala Gly Thr Val Asp Arg Gly GlyAla Ala Asp Leu 100 105 110 Arg Phe Val Pro Leu Cys Arg Arg Arg Tyr ArgIle Leu Glu Ala Asp 115 120 125 Val Thr Asp Arg Asp Pro Gly Glu Ala LeuGlu Ala Val Ile Pro Ala 130 135 140 Thr Ala Ala Met Asp Val Cys Arg ValLeu Leu Thr Gly Glu Ile Gly 145 150 155 160 Glu Pro Gly Ala Asp Leu AlaAsp Leu Glu Arg Arg Tyr Gln Asp Arg 165 170 175 Phe Tyr Ala Leu Asp PheGly Thr Asp Pro Ala Ala Arg Thr Cys Gly 180 185 190 Pro Gly Pro Glu LysThr Ser Met Glu Glu Arg Lys Arg Gln Arg Gly 195 200 205 Arg Ala Ala ArgIle Thr Pro Leu Arg Arg Trp Arg Val Arg Trp Val 210 215 220 Arg Asn Arg225

What is claimed:
 1. An isolated polynucleotide comprising a sequenceselected from the group consisting of: (a) sequences provided in SEQ IDNOs:75, 83, 85, 1-37, 51-74, 76-78 and 86; (b) complements of thesequences provided in SEQ ID NOs: 75, 83, 85, 1-37, 51-74, and 76-78;(c) sequences consisting of at least 20 contiguous residues of asequence provided in SEQ ID NOs: 75, 83, 85, 1-37, 51-74, 76-78 and 87;(d) sequences that hybridize to a sequence provided in SEQ ID NOs: 75,83, 85, 1-37, 51-74, 76-78 and 86, under highly stringent conditions;(e) sequences having at least 75% identity to a sequence of SEQ ID NOs:75, 83, 85, 1-37, 51-74, 76-78 and 87; (f) sequences having at least 90%identity to a sequence of SEQ ID NOs: 75, 83, 85, 1-37, 51-74, 76-78 and87; and (g) degenerate variants of a sequence provided in SEQ ID NOs:75, 83, 85, 1-37, 51-74, 76-78 and
 87. 2. An isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) sequences encoded by a polynucleotide of claim 1; (b) sequenceshaving at least 70% identity to a sequence encoded by a polynucleotideof claim 1; (c) sequences having at least 90% identity to a sequenceencoded by a polynucleotide of claim 1; (d) sequences set forth in SEQID NOs:79, 84, 86, 80-82, 38-50 and 88-89; (e) sequences having at least70% identity to a sequence set forth in SEQ ID NOs: 79, 84, 86, 80-82,38-50 and 88-89; and (f) sequences having at least 90% identity to asequence set forth in SEQ ID NOs: 79, 84, 86, 80-82, 38-50 and 88-89. 3.An expression vector comprising a polynucleotide of claim 1 operablylinked to an expression control sequence.
 4. A host cell transformed ortransfected with an expression vector according to claim
 3. 5. A methodof making a polypeptide comprising: culturing a host cell transformed ortransfected with an expression vector according to claim 3 underconditions whereby the polynucleotide is expressed and the polypeptideis produced; and recovering the polypeptide.
 6. An isolated antibody, orantigen-binding fragment thereof, that specifically binds to apolypeptide of claim
 2. 7. The antibody according to claim 6 whereinsaid antibody is a monoclonal antibody.
 8. The antibody according toclaim 6 wherein said antibody is a human antibody.
 9. The antibodyaccording to claim 6 wherein said antibody has been humanized.
 10. Theantibody according to claim 6 wherein said antibody binds to a flagellinprotein.
 11. The antibody according to claim 10 wherein said antibody isa neutralizing antibody against said flagellin protein.
 12. The antibodyaccording to claim 6 wherein said antibody blocks the interactionbetween a flagellin protein and a Toll-like receptor
 13. The antibodyaccording to claim 12 wherein said Toll-like receptor is TLR5.
 14. Afusion protein comprising at least one polypeptide according to claim 2.15. An oligonucleotide that hybridizes to a polynucleotide according toclaim 1 under highly stringent conditions.
 16. The oligonucleotide ofclaim 15 wherein said polynucleotide according to claim 1 comprises apolynucleotide that encodes a flagellin protein.
 17. A method ofstimulating and/or, expanding T cells specific for an enteric bacterialprotein, comprising contacting T cells with at least one componentselected from the group consisting of: (a) polypeptides according toclaim 2; (b) polynucleotides according to claim 1; and (c)antigen-presenting cells that express a polynucleotide according toclaim 1, under conditions and for a time sufficient to permit thestimulation and/or expansion of T cells.
 18. The method according toclaim 17 wherein said T cells are CD4+ T cells.
 19. The method accordingto claim 18 wherein said CD4+ T cells mediate a decrease in inflammationin the colon.
 20. The method according to claim 17 wherein saidbacterial antigen comprises a flagellin polypeptide.
 21. An isolated Tcell population, comprising T cells prepared according to the method ofclaim
 17. 22. The population of T cells according to claim 19 whereinsaid T cells produce a cytokine selected from the group consisting ofinterleukin 10 (IL-10), interleukin 4 (IL-4), interleukin 5 (IL-5) andtransforming growth factor beta (TGFβ.
 23. The population of T cellsaccording to claim 19 wherein said T cells produce IL-10.
 24. Thepopulation of T cells according to claim 19 wherein said T cells producetransforming growth factor beta (TGFβ.
 25. The population of T cellsaccording to claim 19 wherein said T cells produce IL-10 and TGFβ.
 26. Acomposition comprising a first component selected from the groupconsisting of physiologically acceptable carriers and a second componentselected from the group consisting of: (a) polypeptides according toclaim 2; (b) polynucleotides according to claim 1; (c) antibodiesaccording to claim 5; (d) fusion proteins according to claim 14; (e) Tcell populations according to claim 21; and (f) antigen presenting cellsthat express a polypeptide according to claim
 2. 27. The composition ofclaim 26 further comprising an immunostimulant.
 28. The composition ofclaim 25 wherein said immunostimulant is an adjuvant selected from thegroup consisting of Freund's Incomplete Adjuvant; Freund's CompleteAdjuvant; Merck Adjuvant 65; AS-1, AS-2; aluminum hydroxide gel;aluminum phosphate; a salt of calcium, iron or zinc; an insolublesuspension of acylated tyrosine acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A, QS21, aminoalkyl glucosaminide4-phosphates, and quil A.
 29. A method of detecting the presence ofinflammatory bowel disease in a mammal, comprising the steps of: (a)obtaining a biological sample from the mammal, wherein said biologicalsample comprises antibodies; (b) contacting the biological sample with apolypeptide of claim 2; (c) detecting in the sample an amount ofantibody that binds to the polypeptide; and (d) comparing the amount ofbound antibody to a predetermined cut-off value and therefromdetermining the presence of inflammatory bowel disease in the mammal.30. The method of claim 29 wherein said biological sample is selectedfrom the group consisting of sera, stool, tissue or other materialobtained by colonoscopy, tissue or other material obtained by ileoscopy,tissue or other material obtained by esophagogastroduodenoscopy (EGP),and tissue or other material obtained by surgery.
 31. The method ofclaim 29 wherein said polypeptide of claim 2 comprises a flagellinpolypeptide.
 32. A method of detecting the presence of inflammatorybowel disease in a mammal, comprising the steps of: (a) obtaining abiological sample from said mammal, wherein said biological samplecomprises polynucleotides; (b) contacting said sample with at least oneoligonucleotide that is at least in part complementary to apolynucleotide of claim 1; (c) detecting in the sample an amount of apolynucleotide that hybridizes to said oligonucleotide; and (d)comparing the amount of said polynucleotide that hybridizes to saidoligonucleotide to a predetermined cut-off value, and therefromdetermining the presence of inflammatory bowel disease in a mammal. 33.The method of claim 32 wherein said oligonucleotide hybridizes to apolynucleotide of claim 1 under moderately stringent conditions.
 34. Themethod according to claim 32, wherein the amount of polynucleotide thathybridizes to said oligonucleotide is determined using a polymerasechain reaction.
 35. The method according to claim 32, wherein the amountof polynucleotide that hybridizes to said oligonucleotide is determinedusing a hybridization assay.
 36. The method according to claim 32wherein said biological sample is selected from the group consisting ofsera, stool, tissue or other material obtained by colonoscopy, tissue orother material obtained by ileoscopy, tissue or other material obtainedby esophagogastroduodenoscopy (EGP), and tissue or other materialobtained by surgery.
 37. The method according to claim 32 wherein saidpolynucleotide of claim 1 comprises a polynucleotide that encodes aflagellin protein.
 38. A method of stimulating and/or expanding B cellsthat produce antibodies specific for an enteric bacterial protein,comprising contacting B cells with at least one component selected fromthe group consisting of: (a) polypeptides according to claim 2; and (b)polynucleotides according to claim 1; under conditions and for a timesufficient to permit the stimulation and/or expansion of B cells. 39.The method according to claim 38 wherein said B cells produce antibodiesthat bind a flagellin protein.
 40. The method according to claim 39wherein said antibodies are neutralizing antibodies against saidflagellin protein.
 41. The method according to claim 39 wherein saidantibodies block the interaction between a flagellin protein and aToll-like receptor.
 42. The method according to claim 41 wherein saidToll-like receptor comprises TLR5.
 43. An isolated B cell population,comprising B cells prepared according to any one of the methods ofclaims 39-42.
 44. A method of stimulating an immune response in amammal, comprising administering to the mammal the composition of anyone of claims 26-28.
 45. The method according to claim 44 wherein saidimmune response comprises T cells that produce a cytokine selected fromthe group consisting of interleukin 10 (IL-10), interleukin 4 (IL-4),interleukin 5 (IL-5) and transforming growth factor beta (TGFβ.
 46. Themethod according to claim 45 wherein said cytokine comprises IL-10and/or TGFβ.
 47. A method of decreasing gastrointestinal inflammationassociated with inflammatory bowel disease in a mammal, comprisingadministering to said mammal the composition of any one of claims 26-28.48. A method of identifying bacterial antigens associated withinflammatory bowel disease in a mammal, comprising the steps of: (a)obtaining a biological sample from said mammal wherein said biologicalsample comprises T cells; (b) contacting said biological sample with atleast one component selected from the group consisting of: (i)polypeptides according to claim 2; (ii) polynucleotides according toclaim 1; and (iii) antigen-presenting cells that express apolynucleotide according to claim 1; under conditions and for a timesufficient to permit the stimulation and/or expansion of T cells; (c)detecting in the sample the magnitude of said stimulation and/orexpansion of T cells; and (d) comparing the magnitude of saidstimulation and/or expansion to a predetermined cut-off value, andtherefrom identifying bacterial antigens associated with inflammatorybowel disease in the mammal.
 49. The method of claim 48 wherein saidmammal is a mouse.
 50. The method of claim 49 wherein said mouse is of astrain selected from the group consisting of C3H/HeJ Bir, BALB/cIL-10−/−, B6 IL-10−/−, B10 IL-10−/−, MDR1a −/−, TCRα −/−, IL-2 −/−,IL-2R −/−, mice with DSS (Dextransodiumsulfate) induced colitis, Gα_(ai)−/−, and CD45 RB transgenic mice.
 51. The method of claim 49 whereinsaid mouse is of the C3H/HeJ Bir strain.
 52. A method of monitoring theprogression of inflammatory bowel disease in a mammal, comprising thesteps of: (a) obtaining a biological sample from the mammal, whereinsaid biological sample comprises antibodies; (b) contacting thebiological sample with a polypeptide of claim 2; (c) detecting in thesample an amount of antibody that binds to the polypeptide; and (d)repeating steps (a), (b), and (c) using a biological sample obtainedfrom the mammal at a subsequent point in time; and (e) comparing theamount of bound antibody in step (c) to the amount of bound antibody instep (d) and therefrom monitoring the progression of inflammatory boweldisease in the mammal.
 53. A method of identifying bacterial antigensassociated with inflammatory bowel disease in a first mammal, comprisingthe steps of: (a) obtaining a biological sample from said first mammalwherein said biological sample comprises DNA from cecal bacteria; (b)constructing an expression library with said DNA; (c) screening saidexpression library with sera from either said first mammal or a secondmammal with inflammatory bowel disease; thereby identifying bacterialantigens associated with inflammatory bowel disease.
 54. The method of53 wherein said first and said second mammal are both mice.
 55. Themethod of 53 wherein said first mammal is a C3H/HeJ Bir mouse and saidsecond mammal is a mouse of a strain selected from the group consistingof BALB/c IL-10−/−, B6 IL-10−/−, B10 IL-10 −/−, MDR1a −/−, TCRα −/−,IL-2 −/−, IL-2R −/−, mice with DSS (Dextransodiumsulfate) inducedcolitis, Gα_(ai) −/−, and CD45 RB transgenic mice.
 56. The method of 53wherein said first mammal is a mouse and said second mammal is a human.57. A method of monitoring the progression of inflammatory bowel diseasein a mammal, comprising the steps of: (a) obtaining a biological samplefrom said mammal, wherein said biological sample comprisespolynucleotides; (b) contacting said sample with at least oneoligonucleotide that is at least in part complementary to apolynucleotide of claim 1; (c) detecting in the sample an amount of apolynucleotide that hybridizes to said oligonucleotide; (d) repeatingsteps (a), (b), and (c) using a biological sample obtained from saidmammal at a subsequent point in time; and (e) comparing the amount ofsaid polynucleotide that hybridizes to said oligonucleotide in step (c)to the amount of said polynucleotide that hybridizes to saidoligonucleotide in step (d); and therefrom monitoring the progression ofinflammatory bowel disease in a mammal.
 58. The method of claim 57wherein said oligonucleotide hybridizes to a polynucleotide of claim 1under moderately stringent conditions.
 59. The method according to claim57, wherein the amount of polynucleotide that hybridizes to saidoligonucleotide is determined using a polymerase chain reaction.
 60. Themethod according to claim 57, wherein the amount of polynucleotide thathybridizes to said oligonucleotide is determined using a hybridizationassay.
 61. The method according to claim 57 wherein said biologicalsample is selected from the group consisting of sera, stool, tissue orother material obtained by colonoscopy, tissue or other materialobtained by ileoscopy, tissue or other material obtained byesophagogastroduodenoscopy (EGP), and tissue or other material obtainedby surgery.
 62. The method according to claim 57 wherein saidpolynucleotide of claim 1 comprises a polynucleotide that encodes aflagellin protein.
 63. A diagnostic kit comprising at least oneoligonucleotide according to claim
 15. 64. A diagnostic kit comprisingat least one antibody according to claim 5 and a detection reagent,wherein the detection reagent comprises a reporter group.
 65. Adiagnostic kit, comprising: (a) a portion of at least one or morepolypeptides according to claim 2 wherein said portion can be bound byan antibody; and (b) a detection reagent comprising a reporter group.66. A kit according to claim 65, wherein said portion of at least one ormore polypeptides is immobilized on a solid support.
 67. A kit accordingto claim 65, wherein the detection reagent comprises ananti-immunoglobulin, protein G, protein A, or a lectin.
 68. A kitaccording to claim 65, wherein the reporter group is selected from thegroup consisting of radioactive groups, fluorescent groups, luminescentgroups, enzymes, biotin, and dyes.
 69. An immunogenic compositioncomprising a compound of claim 2, an antigen and a suitable carrier. 70.A method for identifying an inflammatory bowel disease type in apatient, comprising: (a) obtaining an antibody comprising biologicalsample from a patient; (b) contacting the biological sample with apolypeptide of claim 2; (c) detecting in the sample an amount ofantibody that binds to the polypeptide; and (d) comparing the amount ofbound antibody to a predetermined value associated with and therefromsubdividing the inflammatory bowel disease type.
 71. A method of claimclaim 70, wherein the disease type is selected from ulcerative colitisand Crohn's Disease.
 72. The method of claim 70 wherein said polypeptideof claim 2 comprises a flagellin polypeptide.